CN108884447A - Coronavirus (coronavirus) - Google Patents

Coronavirus (coronavirus) Download PDF

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CN108884447A
CN108884447A CN201780006854.2A CN201780006854A CN108884447A CN 108884447 A CN108884447 A CN 108884447A CN 201780006854 A CN201780006854 A CN 201780006854A CN 108884447 A CN108884447 A CN 108884447A
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coronavirus
val
gene
leu
virus
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CN108884447B (en
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E.比克顿
S.基普
P.布里顿
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Pirbright Institute
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Abstract

The present invention provides a live attenuated coronavirus comprising a mutation in the non-structural protein nsp-3 and/or a deletion of the helper proteins 3a and 3 b. The coronaviruses can be used as vaccines for treating and/or preventing diseases in a subject, such as infectious bronchitis.

Description

Coronavirus
Invention field
The present invention relates to the coronavirus of attenuation, and it includes so that virus is had the pathogenic mutation reduced.The present invention is also It is related to purposes of such coronavirus in the vaccine for preventing and/or treating disease.
Background technique
Avian infectious bronchitis virus (IBV), the pathogen of infective bronchitis (IB) are that the height of poultry is infectious And infectious agent, it is mainly replicated in respiratory tract, but also replicated in the epithelial cell of enteron aisle, kidney and fallopian tubal.IBV is Nido virales (Order Nidovirales), coronaviridae (Family Coronaviridae), coronavirus subfamily The member of (Subfamily Coronavirinae) and γ coronavirus genus (Gammacoronavirus)；Genetically unusual phase As coronavirus cause the disease of turkey, guinea fowl and pheasant.
The clinical sign of IB includes sneezing, tracheal rale, nasal mucus and wheezing.There is meat birds reduced weight to increase Add, and the birds that lay eggs descend out less egg and generate the egg of poor quality.Respiratory tract infection makes chicken to Secondary bacterial sexuality Contaminate susceptible, secondary bacterial infection can be chicken fatal.Virus can also cause fallopian tubal permanently to damage Wound, especially in chicken, leads to reduced egg production and quality；And permanent damage is caused to kidney, occasionally resulting in can be with For fatefulue kidney trouble.
It has been reported that IBV economic loss caused by domestic fowl farming will be more than any other infectious disease.Although attenuation Live vaccine and the vaccine of inactivation are commonly used to control IBV, but due to vaccine decompose or introduce it is unrelated with vaccine used new IBV serotype can be lost using vaccine inoculation protection obtained.Risk is caused to domestic fowl farming.
In addition, industrial need to develop the vaccine for being suitable for using in ovum, with improve vaccination program efficiency and at This benefit.Significant challenge relevant to the inoculation of ovum intradermal vaccine is the case where virus allows for the antiviral antibody derived from parent Lower duplication, it is pathogenic without being caused to embryo.Current IBV vaccine is multiple in embryonated egg (embryonated egg) It is obtained after passage, this leads to have the pathogenic virus reduced to chicken, therefore they may be used as the live vaccine of attenuation.So And this viroid almost always shows the virulence increase to embryo, therefore cannot be used for the inoculation of ovum intradermal vaccine, because they cause Reduced hatching rate.See that 70% hatching rate reduces in some cases.
Attenuation after repeatedly passing in embryonated egg also encounters other shortcomings.It is a kind of empirical method, because virus Attenuation is random, and viral passages all can be different every time, therefore is passed to be attenuated purpose by the egg of different series It will lead to a different set of mutation for leading to attenuation for identical virus.There is also efficacy problems relevant to the process：It is some prominent Become the duplication that will affect virus, and some mutation may make viral excessive attenuation.Mutation can also occur in S gene, Immunogenicity can also be influenced, to influence desired immune response, and potential vaccine cannot be directed to desired serotype Protection is provided.Additionally, there are the problems related to recovery virulence and vaccine stability.
It is important that exploitation is for controlling the new and safer vaccine of IBV.Therefore, it is necessary to unrelated with these problems IBV vaccine can be especially useful for the vaccine of ovum intradermal vaccine inoculation.
Summary of the invention
The present inventor is using reverse-genetics approach reasonably to weaken IBV.This method ratio repeatedly passes in embryonated egg Random attenuation after generation is more controllable, because the position of each mutation is known, and can derive its influence to virus, The reason of being attenuated.
Using their reverse-genetics approach, the present inventor, which has identified, causes virus to have the pathogenic water reduced Flat various mutation.Pathogenic level can be reduced, so that can replicate when virus is applied to embryonated egg without right Embryo causes pathogenic.This viroid be applicable to ovum intradermal vaccine inoculation, this be in embryonated egg repeatedly pass on after generate The significant advantage that attenuation IBV vaccine is compared.
Therefore, in a first aspect, it includes nonstructural proteins nsp- the present invention provides the coronavirus of attenuation living The missing of mutation and/or auxiliary protein 3a and/or 3b in 3.
Mutation in nsp-3 can be in adenosine diphosphate-region ribose -1 '-phosphatase (ADRP).
Modified nsp-3 gene can encode the protein comprising one or more amino acid mutations selected from the group below：
a)SEQ ID NO:373rd Asn (N) to Ala (A) in 6；With
b)SEQ ID NO:379th Gly (G) to Ser (S) in 6.
Modified nsp-3 gene can be encoded comprising SEQ ID NO:The 373rd amino acids are mutated Asn (N) extremely in 6 The protein of Ala (A).
Modified nsp-3 gene can be encoded comprising SEQ ID NO:The 379th amino acids are mutated Gly (G) extremely in 6 The protein of Ser (S).
Modified nsp-3 gene may include one or more nucleotide selected from the group below and replace：
A) and with SEQ ID NO:Sequence shown in 5 is compared to the A to G and nucleotide position 1117 at nucleotide position 1116 The A to C at place；With
B) and with SEQ ID NO:Sequence shown in 5 is compared to the G to A at nucleotide position 1138.
The coronavirus of attenuation living may include with SEQ ID NO:The nucleotide position 37 to 384 of sequence shown in 2 Missing.
Coronavirus can be infectious bronchitis virus (IBV).
Coronavirus can be IBV M41.
Coronavirus may include S protein, the S protein at least partially from the IBV serotype in addition to M41.
For example, S1 subunit or entire S protein can come from the IBV serotype in addition to M41.
Coronavirus according to the first aspect of the invention can have causing a disease for reduction compared with wild type coronavirus Property, so that can replicate when virus is applied to embryonated egg and not be pathogenic to embryo.
In second aspect, the present invention provides combine modified nsp-3 gene defined in the first aspect of the present invention.
In the third aspect, the present invention provides by modified nsp-3 gene coding according to the second aspect of the invention Protein.
In fourth aspect, the present invention provides plasmids, and it includes modified nsp-3 according to the second aspect of the invention The modified gene 3 of gene or the missing with auxilin 3a and/or 3b.
At the 5th aspect, the present invention provides the sides for being used to prepare the coronavirus described according to the first aspect of the invention Method comprising following steps：
(i) plasmid transfection according to the fourth aspect of the invention is entered in host cell；
(ii) recombinant virus infection host cell is used, the recombinant virus includes with nsp-3 gene and/or gene 3 The genome of coronavirus strain；
(iii) allow to occur between the corresponding sequence in gene order and the recombinant virus genomes in the plasmid Homologous recombination is to generate modified gene；And
(iv) selection includes the recombinant virus of the modified gene.
Recombinant virus can be vaccinia virus.
Method can also include the following steps：
(v) recombination of the recycling comprising the modified gene is coronal from the DNA of the recombinant virus from step (iv) Virus.
At the 6th aspect, the present invention provides the cells that can generate coronavirus according to the first aspect of the invention.
At the 7th aspect, the present invention provides vaccine, it includes according to the first aspect of the invention coronavirus and medicine Learn acceptable carriers.
In eighth aspect, the present invention provides the method for treating and/or preventing the disease in subject, this method packets Include the step of applying vaccine according to the seventh aspect of the invention to subject.
Other aspects of the present invention provide：
Vaccine according to the seventh aspect of the invention is used to treat and/or prevent the disease in subject.
Coronavirus according to the first aspect of the invention is in preparation for treating and/or preventing the disease in subject Vaccine in purposes.
Disease may be infective bronchitis (IB).
The method of administration of vaccine can be selected from the group：It is injected after eye drops application, intranasal administration, drinking water application, hatching It is injected in ovum.
It can be inoculated with by ovum intradermal vaccine and carry out vaccine inoculation.
The present invention also provides the method for the vaccine of preparation according to the seventh aspect of the invention, this method includes with according to this The step of coronavirus infection of the first aspect of invention cell according to a sixth aspect of the present invention.
Brief description
Fig. 1：For generating the plasmid of rIBV M41K-S-ADRP.
Fig. 2：For generating the plasmid of rIBV M41K-A-ADRP.
Fig. 3：For generating the plasmid of rIBV M41K-del3ab.
Fig. 4：RIBV M41K-S-ADRP, rIBV M41K-A-ADRP or rIBV M41K- are infected compared with M41-K The A of the birds of del3ab) rale；B) comprehensive respiratory symptom (wheezing and rale)；C it) sends out click (Snicking) and D) fine (all bar charts are shown as analogies, rIBV M41K-A-ADRP, rIBV from left to right for knitting (Ciliary activity) M41K-S-ADRP、rIBV M41K-del3ab、M41-K)。
Fig. 5：4th day clinical sign after infection, viral diagnosis and separation summarize.
Fig. 6：6th day clinical sign after infection, viral diagnosis and separation summarize.
Fig. 7：On CK cell compared with wild type M41-K6 and M41-CK EP4 mutant M41K-S-ADRP 4, M41K- The growth kinetics of S-ADRP 2 (repetition of S-ADRP mutation) and M41K-A-ADRP
Fig. 8：On CK cell compared with wild type M41-CK and M41-K mutant M41K-del3ab 10 and M41K- The growth kinetics of del3ab 1
It is described in detail
The present invention provides mutant coronavirus, have what is reduced to cause a disease compared with corresponding wild type coronavirus Property.
Coronavirus
γ coronavirus genus is the animal virus category for belonging to coronaviridae.Coronavirus is with sense single stranded rna base Because organizing the enveloped virus with helical symmetry.
The Genome Size range of coronavirus is about 27 to 32 kilobase, this is most growing up for any known RNA virus It is small.
Coronavirus mainly infects the upper respiratory tract or gastrointestinal tract of mammal and birds.Five to six kinds be currently known are not The same coronavirus strain infection mankind.Cause the most well-known human coronary virus of serious acute respiratory syndrome (SARS) SARS-CoV has unique pathogenesis, because it causes both the infection of the upper respiratory tract and lower respiratory tract infection and can also draw Play gastroenteritis.Middle East respiration syndrome coronavirus (MERS-CoV) can also cause mankind's lower respiratory tract infection.Think coronal disease Poison causes sizable percentage of all common colds in adult humans.
Coronavirus can also cause a series of diseases of livestock and domestic pets, and some of them can be serious and right Aquaculture constitutes a threat to.The economically important coronavirus of domestic animal includes infectious bronchitis virus (IBV), is mainly drawn It plays the respiratory disease of chicken and seriously affects global domestic fowl farming；Pig coronavirus (transmissible gastroenteritis, TGE) and Niu Guanzhuan Virus, they can all lead to the diarrhea in immature animal.For cat coronavirus there are two types of form, cat enteric coronavirus virus is a kind of The little pathogen of clinical meaning, but the spontaneous mutation of this virus can lead to feline infectious peritonitis (FIP), it is a kind of dead with height Die the relevant disease of rate.
There are also two class canine coronavirus (CCoV), cause one kind of slight enterogastric diseases and have been found that cause to breathe One kind of tract disease.Mouse hepatitis virus (MHV) is a kind of coronavirus, leads to the popular mouse disease of high mortality, especially It is in the colony of laboratory mice.
Coronavirus is divided into four groups, as follows：
α
Canine coronavirus (CCoV)
Cat coronavirus (FeCoV)
Human coronary virus 229E (HCoV-229E)
Porcine epidemic diarrhea virus (PEDV)
Infectious gastroenteritis virus (TGEV)
Human coronary virus NL63 (NL or New Haven)
β
Bovine coronavirus (BCoV)
Dog respiratory and enteric coronavirus (CRCoV)-Southeast Asia and Micronesia (Micronesia) are common
Human coronary virus OC43 (HCoV-OC43)
Mouse hepatitis virus (MHV)
Pigs haemagglutinating encephalomyelitis virus (HEV)
Rat coronaviruse (RCV).Rat coronaviruse is very universal in Australian east, by March, 2008/ In April, it finds in natural and wild grinding tooth type of cluster.
(there is no adopted name) (HCoV-HKU1)
SARS (Severe Acute Respiratory Syndrome) coronavirus (SARS-CoV)
Middle East breathing syndrome coronavirus (MERS-CoV)
γ
Infectious bronchitis virus (IBV)
Turkey coronavirus (pullet disease virus)
Pheasant coronavirus
Guinea fowl coronavirus
δ
Bulbul (Bulbul) coronavirus (BuCoV)
Aphtho virus (ThCoV)
Munia coronavirus (MuCoV)
Pig coronavirus (PorCov) HKU15
Recombination coronavirus of the invention can be originated from α coronavirus, such as TGEV；β coronavirus, such as MHV；Or γ- Coronavirus, such as IBV.
As used herein, term " being originated from " means that recombinating coronavirus includes the core essentially identical with wild type coronavirus Nucleotide sequence.For example, recombination coronavirus of the invention and wild type coronavirus sequence can have at least 80%, 85%, 90%, 95%, 98% or 99% identity.
IBV
Avian infectious bronchitis (IB) is a kind of acute and highly infectious chicken respiratory tract disease, is caused great Economic loss.Disease is characterized in that respiratory tract sign, including wheezes, coughs, sneezing, tracheal rale and runny nose.In young chicken In, it may occur however that serious respiratory distress.In laying hen, respiratory distress, ephritis, egg production reduce and internal egg quality Forfeiture with eggshell quality is common.
In broiler chicken, cough and hair quack sound are common clinical signs, are spread rapidly in all birds of premise.Not The disease incidence of the chicken group of vaccine inoculation is 100%.The death rate changes with age, Strain and secondary infection, but be not inoculated with 60% may be up in the chicken group of vaccine.
The first certified IBV serotype is Massachusetts, but in the U.S., in addition to what is initially identified Except Massachusetts type, currently popular several serotypes, including Arkansas and Delaware.
IBV plants of Beaudette are derivative after at least 150 times passages in chicken embryo.IBV Beaudette is for hatching chicken No longer it is pathogenic, but kills embryo rapidly.
H120 is business attenuation IBVMassachusetts serogroup vaccine strain living, by passing on about in egg containing embryo 120 attenuations.H52 is another Massachusetts vaccine, and represents relatively early in H120 development process and slightly more cause The passaged virus (the 52nd generation) of disease.Usually using the vaccine based on H120.
IB QX is the toxicity field separation strains (field isolate) of IBV.It is sometimes referred to as " Chinese QX ", because it Be initially the 1990s mid-term Chinese In The Area of Qingdao break out disease after separate.Virus is just to Europe from that time Continent sprawling.Since two thousand four, (mainly Dutch, but also from Germany, France, Belgium, Denmark and English in West Europe some areas State's report) serious problem of laying eggs is identified in the case where closely similar virus.
The virus separated from Dutch case is accredited as them by the Dutch research institute of Dai Fenteer (Deventer) and is known as The new strains of D388.For China's association from further test, it is 99% similar which show viruses with China QX virus.It is existing Attenuated live QX sample IBV vaccine strain is developed.
IBV is enveloped virus, replicates and contain the single stranded positive-sense rna gene group of non-segmentation in cytoplasm.IBV has The rna gene group of 27.6kb, and contain as all coronavirus that there are four types of structural proteins；Spike glycoprotein (S), membranelle egg White (E), integrated membrane protein (M) and the nuclear shell protein (N) to interact with geneome RNA.
Genome is organized in the following manner：5 ' UTR- polymerase (replicase) gene-structural protein genes (S-E-M-N)- UTR 3'；Wherein UTR is non-translational region (every about 500 nucleotide in IBV).
Lipid envelope includes three kinds of memebrane proteins：S, M and E.IBV S protein is I type glycoprotein, in endoplasmic reticulum oligomerization And it is assembled into the homotrimer being inserted into virosomal membrane by transmembrane domain, and by conjunction with the noncovalent interaction of M albumen. After mixing Coronavirus particles, S protein is responsible for combining the fusion of receptor in target cell and virus and cell membrane.S glycoprotein is by four A domain composition：The signal sequence cut in the synthesis process；Extracellular domain is present in the outside of Virosome particles；It is responsible for S egg The white transmembrane region being anchored in the double-layer of lipoid of Virosome particles；And cytoplasmic tail.
Recombination coronavirus of the invention can be originated from IBV.For example, IBV can be IBV Beaudette, H120, H52, IB QX, D388 or M41.
IBV can be IBV M41.M41 is a kind of prototype Massachusetts blood separated in nineteen forty-one in the U.S. Clear type.It is to be used as the separation strains of pathogenicity laboratory coloring agent in many laboratories in the world, and can be from ATCC (VR-21TM) buy.Several production of vaccine quotient also use attenuation variant as the IBV epidemic disease for being directed to Massachusetts serotype Seedling, the problem of causing the field.The present inventor's selection uses the bacterial strain, because they have worked in the virus for many years, and And because it can get the sequence of whole viral genes group.The M41 separation strains M41-CK that the present inventor uses is suitable in primary chicken kidney (CK) it is grown in cell, therefore, it is considered that being suitble to recycle from the cDNA of complete genome group as infectious virus.It is pathogenic The representative of IBV, thus can analyze cause it is pathogenic forfeiture or reduced mutation.
The genome sequence of IBV M41-CK is with SEQ ID NO:1 provides.
SEQ ID NO:1IBV M41-CK sequence
ACTTAAGATAGATATTAATATATATCTATCACACTAGCCTTGCGCTAGATTTCCAACTTAACAAAACGG ACTTAAATACCTACAGCTGGTCCTCATAGGTGTTCCATTGCAGTGCACTTTAGTGCCCTGGATGGCACCTGGCCACC TGTCAGGTTTTTGTTATTAAAATCTTATTGTTGCTGGTATCACTGCTTGTTTTGCCGTGTCTCACTTTATACATCCG TTGCTTGGGCTACCTAGTATCCAGCGTCCTACGGGCGCCGTGGCTGGTTCGAGTGCGAAGAACCTCTGGTTCATCTA GCGGTAGGCGGGTGTGTGGAAGTAGCACTTCAGACGTACCGGTTCTGTTGTGTGAAATACGGGGTCACCTCCCCCCA CATACCTCTAAGGGCTTTTGAGCCTAGCGTTGGGCTACGTTCTCGCATAAGGTCGGCTATACGACGTTTGTAGGGGG TAGTGCCAAACAACCCCTGAGGTGACAGGTTCTGGTGGTGTTTAGTGAGCAGACATACAATAGACAGTGACAACATG GCTTCAAGCCTAAAACAGGGAGTATCTCCCAAACTAAGGGATGTCATTCTTGTATCCAAAGACATTCCTGAACAACT TTGTGACGCTTTGTTTTTCTATACGTCACACAACCCTAAGGATTACGCTGATGCTTTTGCAGTTAGGCAGAAGTTTG ATCGTAATCTGCAGACTGGGAAACAGTTCAAATTTGAAACTGTGTGTGGTCTCTTCCTCTTGAAGGGAGTTGACAAA ATAACACCTGGCGTCCCAGCAAAAGTCTTAAAAGCCACTTCTAAGTTGGCAGATTTAGAAGACATCTTTGGTGTCTC TCCCTTTGCAAGAAAATATCGTGAACTTTTGAAGACAGCATGCCAGTGGTCTCTTACTGTAGAAACACTGGATGCTC GTGCACAAACTCTTGATGAAATTTTTGACCCTACTGAAATACTTTGGCTTCAGGTGGCAGCAAAAATCCAAGTTTCG GCTATGGCGATGCGCAGGCTTGTTGGAGAAGTAACTGCAAAAGTCATGGATGCTTTGGGCTCAAATATGAGTGCTCT TTTCCAGATTTTTAAACAACAAATAGTCAGAATTTTTCAAAAAGCGCTGGCTATTTTTGAGAATGTGAGTGAATTAC CACAGCGTATTGCAGCACTTAAGATGGCTTTTGCTAAGTGTGCCAAGTCCATTACTGTTGTGGTTATGGAGAGGACT CTAGTTGTTAGAGAGTTCGCAGGAACTTGTCTTGCAAGCATTAATGGTGCTGTTGCAAAATTCTTTGAAGAACTCCC AAATGGTTTCATGGGTGCTAAAATTTTCACTACACTTGCCTTCTTTAGGGAGGCTGCAGTGAAAATTGTGGATAACA TACCAAATGCACCGAGAGGCACTAAAGGGTTTGAAGTCGTTGGTAATGCCAAAGGTACACAAGTTGTTGTGCGTGGC ATGCGAAATGACTTAACACTGCTTGACCAAAAAGCTGAAATTCCTGTGGAGTCAGAAGGTTGGTCTGCAATTTTGGG TGGACATCTTTGCTATGTCTTTAAGAGTGGTGATCGCTTTTACGCGGCACCTCTTTCAGGAAATTTTGCATTGCATG ATGTGCATTGTTGTGAGCGTGTTGTCTGTCTTTCTGATGGTGTAACACCGGAGATAAATGATGGACTTATTCTTGCA GCAATCTACTCTTCTTTTAGTGTCGCAGAACTTGTGGCAGCCATTAAAAGGGGTGAACCATTTAAGTTTCTGGGTCA TAAATTTGTGTATGCAAAGGATGCAGCAGTTTCTTTTACATTAGCGAAGGCTGCTACTATTGCAGATGTTTTGAAGC TGTTTCAATCAGCGCGTGTGAAAGTAGAAGATGTTTGGTCTTCACTTACTGAAAAGTCTTTTGAATTCTGGAGGCTT GCATATGGAAAAGTGCGTAATCTCGAAGAATTTGTTAAGACTTGTTTTTGTAAGGCTCAAATGGCGATTGTGATTTT AGCGACAGTGCTTGGAGAGGGCATTTGGCATCTTGTTTCGCAAGTCATCTATAAAGTAGGTGGTCTTTTTACTAAAG TTGTTGACTTTTGTGAAAAATATTGGAAAGGTTTTTGTGCACAGTTGAAAAGAGCTAAGCTCATTGTCACTGAAACC CTCTGTGTTTTGAAAGGAGTTGCACAGCATTGTTTTCAACTATTGCTGGATGCAATACAGTTTATGTATAAAAGTTT TAAGAAGTGTGCACTTGGTAGAATCCATGGAGACTTGCTCTTCTGGAAAGGAGGTGTGCACAAAATTATTCAAGAGG GCGATGAAATTTGGTTTGACGCCATTGATAGTATTGATGTTGAAGATCTGGGTGTTGTTCAAGAAAAATTGATTGAT TTTGATGTTTGTGATAATGTGACACTTCCAGAGAACCAACCCGGTCATATGGTTCAAATCGAGGATGACGGAAAGAA CTACATGTTCTTCCGCTTCAAAAAGGATGAGAACATTTATTATACACCAATGTCACAGCTTGGTGCTATTAATGTGG TTTGCAAAGCAGGCGGTAAAACTGTCACCTTTGGAGAAACTACTGTGCAAGAAATACCACCACCTGATGTTGTGTTT ATTAAGGTTAGCATTGAGTGTTGTGGTGAACCATGGAATACAATCTTCAAAAAGGCTTATAAGGAGCCCATTGAAGT AGAGACAGACCTCACAGTTGAACAATTGCTCTCTGTGGTCTATGAGAAAATGTGTGATGATCTCAAGCTGTTTCCGG AGGCTCCAGAACCACCACCATTTGAGAATGTCACACTTGTTGATAAGAATGGTAAAGATTTGGATTGCATAAAATCA TGCCATCTGATCTATCGTGATTATGAGAGCGATGATGACATCGAGGAAGAAGATGCAGAAGAATGTGACACGGATTC AGGTGATGCTGAGGAGTGTGACACTAATTCAGAATGTGAAGAAGAAGATGAGGATACTAAAGTGTTGGCTCTTATAC AAGACCCGGCAAGTAACAAATATCCTCTGCCTCTTGATGATGATTATAGCGTCTACAATGGATGTATTGTTCATAAG GACGCTCTCGATGTTGTGAATTTACCATCTGGTGAAGAAACCTTTGTTGTCAATAACTGCTTTGAAGGGGCTGTTAA AGCTCTTCCGCAGAAAGTTATTGATGTTCTAGGTGACTGGGGTGAGGCTGTTGATGCGCAAGAACAATTGTGTCAAC AAGAATCAACTCGGGTCATATCTGAGAAATCAGTTGAGGGTTTTACTGGTAGTTGTGATGCAATGGCTGAACAAGCT ATTGTTGAAGAGCAGGAAATAGTACCTGTTGTTGAACAAAGTCAGGATGTAGTTGTTTTTACACCTGCAGACCTAGA AGTTGTTAAAGAAACAGCAGAAGAGGTTGATGAGTTTATTCTCATTTCTGCTGTCCCTAAAGAAGAAGTTGTGTCTC AGGAGAAAGAGGAGCCACAGGTTGAGCAAGAGCCTACCCTAGTTGTTAAAGCACAACGTGAGAAGAAGGCTAAAAAG TTCAAAGTTAAACCAGCTACATGTGAAAAACCCAAATTTTTGGAGTACAAAACATGTGTGGGTGATTTGGCTGTTGT AATTGCCAAAGCATTGGATGAGTTTAAAGAGTTCTGCATTGTAAACGCTGCAAATGAGCACATGTCGCATGGTGGTG GCGTTGCAAAGGCAATTGCAGACTTTTGTGGACCGGACTTTGTTGAATATTGCGCGGACTATGTTAAGAAACATGGT CCACAGCAAAAACTTGTCACACCTTCATTTGTTAAAGGCATTCAATGTGTGAATAATGTTGTAGGACCTCGCCATGG AGACAGCAACTTGCGTGAGAAGCTTGTTGCTGCTTACAAGAGTGTTCTTGTAGGTGGAGTGGTTAACTATGTTGTGC CAGTTCTCTCATCAGGGATTTTTGGTGTAGATTTTAAAATATCAATAGATGCTATGCGCGAAGCTTTTAAAGGTTGT GCCATACGCGTTCTTTTATTTTCTCTGAGTCAAGAACACATCGATTATTTCGATGCAACTTGTAAGCAGAAGACAAT TTATCTTACGGAGGATGGTGTTAAATACCGCTCTGTTGTTTTAAAACCTGGTGATTCTTTGGGTCAATTTGGACAGG TTTTTGCAAGAAATAAGGTAGTCTTTTCGGCTGATGATGTTGAGGATAAAGAAATCCTCTTTATACCCACAACTGAC AAGACTATTCTTGAATATTATGGTTTAGATGCGCAAAAGTATGTAACATATTTGCAAACGCTTGCGCAGAAATGGGA TGTTCAATATAGAGACAATTTTGTTATATTAGAGTGGCGTGACGGAAATTGCTGGATTAGTTCAGCAATAGTTCTCC TTCAAGCTGCTAAAATTAGATTTAAAGGTTTTCTTGCAGAAGCATGGGCTAAACTGTTGGGTGGAGATCCTACAGAC TTTGTTGCCTGGTGTTATGCAAGTTGCAATGCTAAAGTAGGTGATTTTTCAGATGCTAATTGGCTTTTGGCCAATTT AGCAGAACATTTTGACGCAGATTACACAAATGCACTTCTTAAGAAGTGTGTGTCGTGCAATTGTGGTGTTAAGAGTT ATGAACTTAGGGGTCTTGAAGCCTGTATTCAGCCAGTTCGAGCACCTAATCTTCTACATTTTAAAACGCAATATTCA AATTGCCCAACCTGTGGTGCAAGTAGTACGGATGAAGTAATAGAAGCTTCATTACCGTACTTATTGCTTTTTGCTAC TGATGGTCCTGCTACAGTTGATTGTGATGAAAATGCTGTAGGGACTGTTGTTTTCATTGGCTCTACTAATAGTGGCC ATTGTTATACACAAGCCGATGGTAAGGCTTTTGACAATCTTGCTAAGGATAGAAAATTTGGAAGGAAGTCGCCTTAC ATTACAGCAATGTATACACGTTTTTCTCTTAGGAGTGAAAATCCCCTACTTGTTGTTGAACATAGTAAGGGTAAAGC TAAAGTAGTAAAAGAAGATGTTTCTAACCTTGCTACTAGTTCTAAAGCCAGTTTTGACGATCTTACTGACTTTGAAC AGTGGTATGATAGCAACATCTATGAGAGTCTTAAAGTGCAGGAGACACCTGATAATCTTGATGAATATGTGTCATTT ACGACAAAGGAAGATTCTAAGTTGCCACTGACACTTAAAGTTAGAGGTATCAAATCAGTTGTTGACTTTAGGTCTAA GGATGGTTTTACTTATAAGTTAACACCTGATACTGATGAAAATTCAAAAACACCAGTCTACTACCCAGTCTTGGATT CTATTAGTCTTAGGGCAATATGGGTTGAAGGCAGTGCTAATTTTGTTGTTGGGCATCCAAATTATTATAGTAAGTCT CTCCGAATTCCCACGTTTTGGGAAAATGCCGAGAGCTTTGTTAAAATGGGTTATAAAATTGATGGTGTAACTATGGG CCTTTGGCGTGCAGAACACCTTAATAAACCTAATTTGGAGAGAATTTTTAACATTGCTAAGAAAGCTATTGTTGGAT CTAGTGTTGTTACTACGCAGTGTGGTAAAATACTAGTTAAAGCAGCTACATACGTTGCCGATAAAGTAGGTGATGGT GTAGTTCGCAATATTACAGATAGAATTAAGGGTCTTTGTGGATTCACACGTGGCCATTTTGAAAAGAAAATGTCCCT ACAATTTCTAAAGACACTTGTGTTCTTTTTCTTTTATTTCTTAAAGGCTAGTGCTAAGAGTTTAGTTTCTAGCTATA AGATTGTGTTATGTAAGGTGGTGTTTGCTACCTTACTTATAGTGTGGTTTATATACACAAGTAATCCAGTAGTGTTT ACTGGAATACGTGTGCTAGACTTCCTATTTGAAGGTTCTTTATGTGGTCCTTATAATGACTACGGTAAAGATTCTTT TGATGTGTTACGCTATTGTGCAGGTGATTTTACTTGTCGTGTGTGTTTACATGATAGAGATTCACTTCATCTGTACA AACATGCTTATAGCGTAGAACAAATTTATAAGGATGCAGCTTCTGGCATTAACTTTAATTGGAATTGGCTTTATTTG GTCTTTCTAATATTATTTGTTAAGCCAGTGGCAGGTTTTGTTATTATTTGTTATTGTGTTAAGTATTTGGTATTGAG TTCAACTGTGTTGCAAACTGGTGTAGGTTTTCTAGATTGGTTTGTAAAAACAGTTTTTACCCATTTTAATTTTATGG GAGCGGGATTTTATTTCTGGCTCTTTTACAAGATATACGTACAAGTGCATCATATATTGTACTGTAAGGATGTAACA TGTGAAGTGTGCAAGAGAGTTGCACGCAGCAACAGGCAAGAGGTTAGCGTTGTAGTTGGTGGACGCAAGCAAATAGT GCATGTTTACACTAATTCTGGCTATAACTTTTGTAAGAGACATAATTGGTATTGTAGAAATTGTGATGATTATGGTC ACCAAAATACATTTATGTCCCCTGAAGTTGCTGGCGAGCTTTCTGAAAAGCTTAAGCGCCATGTTAAACCTACAGCA TATGCTTACCACGTTGTGTATGAGGCATGCGTGGTTGATGATTTTGTTAATTTAAAATATAAGGCTGCAATTCCTGG TAAGGATAATGCATCTTCTGCTGTTAAGTGTTTCAGTGTTACAGATTTTTTAAAGAAAGCTGTTTTTCTTAAGGAGG CATTGAAATGTGAACAAATATCTAATGATGGTTTTATAGTGTGTAATACACAGAGTGCGCATGCACTAGAGGAAGCA AAGAATGCAGCCGTCTATTATGCGCAATATCTGTGTAAGCCAATACTTATACTTGACCAGGCACTTTATGAGCAATT AATAGTAGAGCCTGTGTCTAAGAGTGTTATAGATAAAGTGTGTAGCATTTTGTCTAATATAATATCTGTAGATACTG CAGCTTTAAATTATAAGGCAGGCACACTTCGTGATGCTCTGCTTTCTATTACTAAAGACGAAGAAGCCGTAGATATG GCTATCTTCTGCCACAATCATGAAGTGGAATACACTGGTGACGGTTTTACTAATGTGATACCGTCATATGGTATGGA CACTGATAAGTTGACACCTCGTGATAGAGGGTTTTTGATAAATGCAGATGCTTCTATTGCTAATTTAAGAGTCAAAA ATGCTCCTCCGGTAGTATGGAAGTTTTCTGATCTTATTAAATTGTCTGACAGTTGCCTTAAATATTTAATTTCAGCT ACTGTCAAGTCAGGAGGTCGTTTCTTTATAACAAAGTCTGGTGCTAAACAAGTTATTTCTTGTCATACCCAGAAACT GTTGGTAGAGAAAAAGGCAGGTGGTGTTATTAATAACACTTTTAAATGGTTTATGAGTTGTTTTAAATGGCTTTTTG TCTTTTATATACTTTTTACAGCATGTTGTTTGGGTTACTACTATATGGAGATGAATAAAAGTTTTGTTCACCCCATG TATGATGTAAACTCCACACTGCATGTTGAAGGGTTCAAAGTTATAGACAAAGGTGTTATTAGAGAGATTGTGTCAGA AGATAATTGTTTCTCTAATAAGTTTGTTAATTTTGACGCCTTTTGGGGTAAATCATATGAAAATAATAAAAACTGTC CAATTGTTACAGTTGTTATAGATGGTGACGGGACAGTAGCTGTTGGTGTTCCTGGTTTTGTATCATGGGTTATGGAT GGTGTTATGTTTGTGCATATGACACAGACTGATCGTAGACCTTGGTACATTCCTACCTGGTTTAATAGAGAAATTGT TGGTTACACTCAGGATTCAATTATCACTGAGGGTAGTTTTTATACATCTATAGCATTATTTTCTGCTAGATGTTTAT ATTTAACAGCCAGCAATACACCTCAATTGTATTGTTTTAATGGCGACAATGATGCACCTGGAGCCTTACCATTTGGT AGTATTATTCCTCATAGAGTATACTTCCAACCTAATGGTGTTAGGCTTATAGTTCCACAACAAATACTGCATACACC CTACATAGTGAAGTTTGTTTCAGACAGCTATTGTAGAGGTAGTGTATGTGAGTATACTAAACCAGGTTACTGTGTGT CACTAGACTCCCAATGGGTTTTGTTTAATGATGAATACATTAGTAAACCTGGCGTTTTCTGTGGTTCTACTGTTAGA GAACTTATGTTTAATATGGTTAGTACATTCTTTACTGGTGTCAACCCTAATATTTATATTCAGCTAGCAACTATGTT TTTAATACTAGTTGTTATTGTGTTAATTTTTGCAATGGTTATAAAGTTTCAAGGTGTTTTTAAAGCTTATGCGACCA TTGTGTTTACAATAATGTTAGTTTGGGTTATTAATGCATTTGTTTTGTGTGTACATAGTTATAATAGTGTTTTAGCT GTTATATTATTAGTACTCTATTGCTATGCATCATTGGTTACAAGTCGCAATACTGCTATAATAATGCATTGTTGGCT TGTTTTTACCTTTGGTTTAATAGTACCCACATGGTTGGCTTGTTGCTATCTGGGATTTATTCTTTATATGTACACAC CGTTGGTTTTCTGGTGTTACGGTACTACTAAAAATACTCGTAAGTTGTATGATGGCAACGAGTTTGTTGGTAATTAT GACCTTGCTGCGAAGAGCACTTTTGTTATTCGTGGTACTGAATTTGTTAAGCTTACGAATGAGATAGGTGATAAATT TGAAGCCTATCTTTCTGCGTATGCTAGACTTAAATACTATTCAGGCACTGGTAGTGAGCAAGATTACTTGCAAGCTT GTCGTGCATGGTTAGCTTATGCTTTGGACCAATATAGAAATAGTGGTGTTGAGGTTGTTTATACCCCACCGCGTTAC TCTATTGGTGTTAGTAGACTACACGCTGGTTTTAAAAAACTAGTTTCTCCTAGTAGTGCTGTTGAGAAGTGCATTGT TAGTGTCTCTTATAGAGGCAATAATCTTAATGGACTGTGGCTGGGTGATTCTATTTACTGCCCACGCCATGTGTTAG GTAAGTTTAGTGGTGACCAGTGGGGTGACGTACTAAACCTTGCTAATAATCATGAGTTTGAAGTTGTAACTCAAAAT GGTGTTACTTTGAATGTTGTCAGCAGGCGGCTTAAAGGAGCAGTTTTAATTTTACAAACTGCAGTTGCCAATGCTGA AACTCCTAAGTATAAGTTTGTTAAAGCTAATTGTGGTGATAGTTTCACTATAGCTTGTTCTTATGGTGGTACAGTTA TAGGACTTTACCCTGTCACTATGCGTTCTAATGGTACTATTAGAGCATCTTTCCTAGCAGGAGCCTGTGGCTCAGTT GGTTTTAATATAGAAAAGGGTGTAGTTAATTTCTTTTATATGCACCATCTTGAGTTACCTAATGCATTACACACTGG AACTGACCTAATGGGTGAGTTTTATGGTGGTTATGTAGATGAAGAGGTTGCGCAAAGAGTGCCACCAGATAATCTAG TTACTAACAATATTGTAGCATGGCTCTATGCGGCAATTATTAGTGTTAAAGAAAGTAGTTTTTCACAACCTAAATGG TTGGAGAGTACTACTGTTTCTATTGAAGATTACAATAGGTGGGCTAGTGATAATGGTTTTACTCCATTTTCCACTAG TACTGCTATTACTAAATTAAGTGCTATAACTGGGGTTGATGTTTGTAAACTCCTTCGCACTATTATGGTAAAAAGTG CTCAATGGGGTAGTGATCCCATTTTAGGACAATATAATTTTGAAGACGAATTGACACCAGAATCTGTATTTAATCAA GTTGGTGGTGTTAGGTTACAGTCTTCTTTTGTAAGAAAAGCTACATCTTGGTTTTGGAGTAGATGTGTATTAGCTTG CTTCTTGTTTGTGTTGTGTGCTATTGTCTTATTTACGGCAGTGCCACTTAAGTTTTATGTACATGCAGCTGTTATTT TGTTGATGGCTGTGCTCTTTATTTCTTTTACTGTTAAACATGTTATGGCATACATGGACACTTTCCTATTGCCTACA TTGATTACAGTTATTATTGGAGTTTGTGCTGAAGTCCCTTTCATATACAATACTCTAATTAGTCAAGTTGTTATTTT CTTAAGCCAATGGTATGATCCTGTAGTCTTTGATACTATGGTACCATGGATGTTATTGCCATTAGTGTTGTACACTG CTTTTAAGTGTGTACAAGGCTGCTATATGAATTCTTTCAATACTTCTTTGTTAATGCTGTATCAGTTTATGAAGTTA GGTTTTGTTATTTACACCTCTTCAAACACTCTTACTGCATATACAGAAGGTAATTGGGAGTTATTCTTTGAGTTGGT TCACACTATTGTGTTGGCTAATGTTAGTAGTAATTCCTTAATTGGTTTAATTGTTTTTAAGTGTGCTAAGTGGATTT TATATTATTGCAATGCAACATACTTTAATAATTATGTGTTAATGGCAGTCATGGTTAATGGCATAGGCTGGCTTTGC ACCTGTTACTTTGGATTGTATTGGTGGGTTAATAAAGTTTTTGGTTTAACCTTAGGTAAATACAATTTTAAAGTTTC AGTAGATCAATATAGGTATATGTGTTTGCATAAGGTAAATCCACCTAAAACTGTGTGGGAGGTCTTTACTACAAATA TACTTATACAAGGAATTGGAGGCGATCGTGTGTTGCCTATAGCTACAGTGCAATCTAAATTGAGTGATGTAAAGTGT ACAACTGTTGTTTTAATGCAGCTTTTGACTAAGCTTAATGTTGAAGCAAATTCAAAAATGCATGCTTATCTTGTTGA GTTACACAATAAAATCCTCGCATCTGATGATGTTGGAGAGTGCATGGATAATTTATTGGGTATGCTTATAACACTAT TTTGTATAGATTCTACTATTGATTTGGGTGAGTATTGTGATGATATACTTAAGAGGTCAACTGTATTACAATCGGTT ACTCAAGAGTTTTCGCACATACCCTCGTATGCTGAATATGAAAGAGCTAAGAGTATTTATGAAAAGGTTTTAGCCGA TTCTAAAAATGGTGGTGTAACACAGCAAGAGCTTGCTGCATATCGTAAAGCTGCCAATATTGCAAAGTCAGTTTTTG ATAGAGACTTGGCTGTTCAAAAGAAGTTAGATAGCATGGCAGAACGTGCTATGACAACAATGTATAAAGAGGCGCGT GTAACTGATAGAAGAGCAAAATTAGTTTCATCATTACATGCACTACTTTTTTCAATGCTTAAGAAAATAGATTCTGA GAAGCTTAATGTCTTATTTGACCAGGCGAATAGTGGTGTTGTACCCCTAGCAACTGTTCCAATTGTTTGTAGTAATA AGCTTACCCTTGTTATACCAGACCCAGAGACGTGGGTCAAGTGTGTGGAGGGTGTGCATGTTACATATTCAACAGTT GTTTGGAATATAGACTGTGTTACTGATGCCGATGGCACAGAGTTACACCCCACTTCTACAGGTAGTGGATTGACTTA CTGTATAAGTGGTGATAATATAGCATGGCCTTTAAAGGTTAACTTGACTAGGAATGGGCATAATAAGGTTGATGTTG CCTTGCAAAATAATGAGCTTATGCCTCACGGTGTAAAGACAAAGGCTTGCGTAGCAGGTGTAGATCAAGCACATTGT AGCGTTGAGTCTAAATGTTATTATACAAGTATTAGTGGCAGTTCAGTTGTAGCTGCTATTACCTCTTCAAATCCTAA TCTGAAAGTAGCCTCTTTTTTGAATGAGGCAGGTAATCAGATTTATGTAGACTTAGACCCACCATGTAAATTTGGTA TGAAAGTGGGTGATAAGGTTGAAGTTGTTTACCTGTATTTTATAAAAAATACGAGGTCTATTGTAAGAGGTATGGTA CTTGGTGCTATATCTAATGTTGTTGTGTTACAATCTAAAGGTCATGAGACAGAGGAAGTGGATGCTGTAGGCATTCT CTCACTTTGTTCTTTTGCAGTAGATCCTGCGGATACATATTGTAAATATGTGGCAGCAGGTAATCAACCTTTAGGTA ACTGTGTTAAAATGTTGACAGTACATAATGGTAGTGGTTTTGCAATAACATCAAAGCCAAGTCCAACTCCGGATCAG GATTCTTATGGAGGAGCTTCTGTGTGTCTTTATTGTAGAGCACATATAGCACACCCTGGCGGAGCAGGAAATTTAGA TGGACGCTGTCAATTTAAAGGTTCTTTTGTGCAAATACCTACTACGGAGAAAGATCCTGTTGGATTCTGTCTACGTA ACAAGGTTTGCACTGTTTGTCAGTGTTGGATTGGTTATGGATGTCAGTGTGATTCACTTAGACAACCTAAACCTTCT GTTCAGTCAGTTGCTGTTGCATCTGGTTTTGATAAGAATTATTTAAACGGGTACGGGGTAGCAGTGAGGCTCGGCTG ATACCCCTAGCTAATGGATGTGACCCCGATGTTGTAAAGCGAGCCTTTGATGTTTGTAATAAGGAATCAGCCGGTAT GTTTCAAAATTTGAAGCGTAACTGTGCACGATTCCAAGAAGTACGTGATACTGAAGATGGAAATCTTGAGTATTGTG ATTCTTATTTTGTGGTTAAACAAACCACTCCTAGTAATTATGAACATGAGAAAGCTTGTTATGAAGACTTAAAGTCA GAAGTAACAGCTGATCATGATTTCTTTGTGTTCAATAAGAACATTTATAATATTAGTAGGCAGAGGCTTACTAAGTA TACTATGATGGATTTTTGCTATGCTTTGCGGCACTTTGACCCAAAGGATTGCGAAGTTCTTAAAGAAATACTTGTCA CTTATGGTTGTATAGAAGATTATCACCCTAAGTGGTTTGAAGAGAATAAGGATTGGTACGACCCAATAGAAAACCCT AAATATTATGCCATGTTGGCTAAAATGGGACCTATTGTACGACGTGCTTTATTGAATGCTATTGAGTTCGGAAACCT CATGGTTGAAAAAGGTTATGTTGGTGTTATTACACTTGATAACCAAGATCTTAATGGCAAATTTTATGATTTTGGTG ATTTTCAGAAGACAGCGCCTGGTGCTGGTGTTCCTGTTTTTGATACGTATTATTCTTACATGATGCCCATCATAGCC ATGACTGATGCGTTGGCACCTGAGAGGTATTTTGAATATGATGTGCATAAGGGTTATAAATCTTATGATCTCCTCAA GTATGATTATACTGAGGAGAAACAAGATTTGTTTCAGAAGTACTTTAAGTATTGGGATCAAGAGTATCACCCTAACT GTCGCGACTGTAGTGATGACAGGTGTTTGATACATTGTGCAAACTTCAACATCTTGTTTTCTACACTTGTACCGCAG ACTTCTTTCGGTAATTTGTGTAGAAAGGTTTTTGTTGATGGTGTACCATTTATAGCTACTTGTGGCTATCATTCTAA GGAACTTGGTGTTATTATGAATCAAGATAACACCATGTCATTTTCAAAAATGGGTTTGAGTCAACTCATGCAGTTTG TTGGAGATCCTGCCTTGTTAGTGGGGACATCCAATAAATTAGTGGATCTTAGAACGTCTTGTTTTAGTGTTTGTGCT TTAGCGTCTGGTATTACTCATCAAACGGTAAAACCAGGTCACTTTAACAAGGATTTCTACGATTTTGCAGAGAAGGC TGGTATGTTTAAGGAAGGTTCTTCTATACCACTTAAACATTTCTTCTACCCACAGACTGGTAATGCTGCTATAAACG ATTATGATTATTATCGTTATAACAGGCCTACCATGTTTGATATACGTCAACTTTTATTTTGTTTAGAAGTGACTTCT AAATATTTTGAATGTTATGAAGGCGGCTGTATACCAGCAAGCCAAGTTGTAGTTAACAATTTAGATAAGAGTGCAGG TTATCCGTTCAATAAGTTTGGAAAGGCCCGTCTCTATTATGAAATGAGTCTAGAGGAGCAGGACCAACTCTTTGAGA GTACAAAGAAGAACGTCCTGCCTACTATAACTCAGATGAATTTAAAATATGCCATATCCGCGAAAAATAGAGCGCGT ACAGTGGCAGGTGTGTCTATCCTTTCTACTATGACTAATAGGCAGTTTCATCAGAAGATTCTTAAGTCTATAGTCAA CACTAGAAACGCTCCTGTAGTTATTGGAACAACCAAGTTTTATGGCGGTTGGGATAACATGTTGAGAAACCTTATTC AGGGTGTTGAAGACCCGATTCTTATGGGTTGGGATTATCCAAAGTGTGATAGAGCAATGCCTAATTTGTTGCGTATA GCAGCATCTTTAGTACTCGCTCGTAAACACACTAATTGTTGTACTTGGTCTGAACGCGTTTATAGGTTGTATAATGA ATGCGCTCAGGTTTTATCTGAAACTGTCTTAGCTACAGGTGGTATATATGTGAAACCTGGTGGTACTAGCAGTGGAG ATGCTACTACTGCTTATGCAAACAGTGTTTTCAACATAATACAAGCCACATCTGCTAATGTTGCGCGTCTTTTGAGT GTTATAACGCGTGATATTGTATATGATGACATTAAGAGCTTGCAGTATGAATTGTACCAGCAGGTTTATAGGCGAGT CAATTTTGACCCAGCATTTGTTGAAAAGTTTTATTCTTATTTGTGTAAGAATTTCTCATTGATGATCTTGTCTGACG ACGGTGTTGTTTGTTATAACAACACATTAGCCAAACAAGGTCTTGTAGCAGATATTTCTGGTTTTAGAGAAGTTCTC TACTATCAGAACAATGTTTTTATGGCTGATTCTAAATGTTGGGTTGAACCAGATTTAGAAAAAGGCCCACATGAATT TTGTTCACAGCACACAATGTTAGTGGAGGTTGATGGTGAGCCTAGATACTTGCCATATCCAGACCCATCACGTATTT TGTGTGCATGTGTTTTTGTAGATGATTTGGATAAGACAGAATCTGTGGCTGTTATGGAGCGTTATATCGCTCTTGCC ATAGATGCGTACCCACTAGTACATCATGAAAATGAGGAGTACAAGAAGGTATTCTTTGTGCTTCTTTCATACATCAG AAAACTCTATCAAGAGCTTTCTCAGAATATGCTTATGGACTACTCTTTTGTAATGGATATAGATAAGGGTAGTAAAT TTTGGGAACAGGAGTTCTATGAAAATATGTATAGAGCCCCTACAACATTACAGTCTTGTGGCGTTTGTGTAGTGTGT AATAGTCAAACTATATTGCGCTGTGGTAATTGTATTCGCAAACCATTTTTGTGTTGTAAGTGTTGCTATGACCATGT CATGCACACAGACCACAAAAATGTTTTGTCTATAAATCCTTACATTTGCTCACAGCCAGGTTGTGGTGAAGCAGATG TTACTAAATTGTACCTCGGAGGTATGTCATACTTCTGCGGTAATCATAAACCAAAGTTATCAATACCGTTAGTATCT AATGGTACAGTGTTTGGAATTTACAGGGCTAATTGTGCAGGTAGCGAAAATGTTGATGATTTTAATCAACTAGCTAC TACTAATTGGTCTACTGTGGAACCTTATATTTTGGCAAATCGTTGTGTAGATTCGTTGAGACGCTTTGCTGCAGAGA CAGTAAAAGCTACAGAAGAATTACATAAGCAACAATTTGCTAGTGCAGAAGTGAGAGAAGTACTCTCAGATCGTGAA TTGATTCTGTCTTGGGAGCCAGGTAAAACCAGGCCTCCATTGAATAGAAATTATGTTTTCACTGGCTTTCACTTTAC TAGAACTAGTAAAGTTCAGCTCGGTGATTTTACATTTGAAAAAGGTGAAGGTAAGGACGTTGTCTATTATCGAGCGA CGTCTACTGCTAAATTGTCTGTTGGAGACATTTTTGTTTTAACCTCACACAATGTTGTTTCTCTTATAGCGCCAACG TTGTGTCCTCAGCAAACCTTTTCTAGGTTTGTGAATTTAAGACCTAATGTGATGGTACCTGCGTGTTTTGTAAATAA CATTCCATTGTACCATTTAGTAGGCAAGCAGAAGCGTACTACAGTACAAGGCCCTCCTGGCAGTGGTAAATCCCATT TTGCTATAGGATTGGCGGCTTACTTTAGTAACGCCCGTGTCGTTTTTACTGCATGCTCTCATGCAGCTGTTGATGCT TTATGTGAAAAAGCTTTTAAGTTTCTTAAAGTAGATGATTGCACTCGTATAGTACCTCAAAGGACTACTATCGATTG CTTCTCTAAGTTTAAAGCTAATGACACAGGCAAAAAGTACATTTTTAGTACTATTAATGCCTTGCCAGAAGTTAGTT GTGACATTCTTTTGGTTGACGAGGTTAGTATGTTGACCAATTACGAATTGTCTTTTATTAATGGTAAGATAAACTAT CAATATGTTGTGTATGTAGGTGATCCTGCTCAATTACCGGCGCCTCGTACGTTGCTTAACGGTTCACTCTCTCCAAA GGATTATAATGTTGTCACAAACCTTATGGTTTGTGTTAAACCTGACATTTTCCTTGCAAAGTGTTACCGTTGTCCTA AAGAAATTGTAGATACTGTTTCTACTCTTGTATATGATGGAAAGTTTATTGCAAATAACCCGGAATCACGTCAGTGT TTCAAGGTTATAGTTAATAATGGTAATTCTGATGTAGGACATGAAAGTGGCTCAGCCTACAACATAACTCAATTAGA ATTTGTGAAAGATTTTGTCTGTCGCAATAAGGAATGGCGGGAAGCAACATTCATTTCACCTTATAATGCTATGAACC AGAGAGCCTACCGTATGCTTGGACTTAATGTTCAGACAGTAGACTCGTCTCAAGGTTCGGAGTATGATTATGTTATC TTTTGTGTTACTGCAGATTCGCAGCATGCACTGAATATTAACAGATTCAATGTAGCGCTTACAAGAGCCAAGCGTGG TATACTAGTTGTCATGCGTCAGCGTGATGAACTATATTCAGCTCTTAAGTTTATAGAGCTTGATAGTGTAGCAAGTC TGCAAGGTACAGGCTTGTTTAAAATTTGCAACAAAGAGTTTAGTGGTGTTCACCCAGCTTATGCAGTCACAACTAAG GCTCTTGCTGCAACTTATAAAGTTAATGATGAACTTGCTGCACTTGTTAACGTGGAAGCTGGTTCAGAAATAACATA TAAACATCTTATTTCTTTGTTAGGGTTTAAGATGAGTGTTAATGTTGAAGGCTGCCACAACATGTTTATAACACGTG ATGAGGCTATCCGCAACGTAAGAGGTTGGGTAGGTTTTGATGTAGAAGCAACACATGCTTGCGGTACTAACATTGGT ACTAACCTGCCTTTCCAAGTAGGTTTCTCTACTGGTGCAGACTTTGTAGTTACGCCTGAGGGACTTGTAGATACTTC AATAGGCAATAATTTTGAGCCTGTGAATTCTAAAGCACCTCCAGGTGAACAATTTAATCACTTGAGAGCGTTATTCA AAAGTGCTAAACCTTGGCATGTTGTAAGGCCAAGGATTGTGCAAATGTTAGCGGATAACCTGTGCAACGTTTCAGAT TGTGTAGTGTTTGTCACGTGGTGTCATGGCCTAGAACTAACCACTTTGCGCTATTTTGTTAAAATAGGCAAGGACCA AGTTTGTTCTTGCGGTTCTAGAGCAACAACTTTTAATTCTCATACTCAGGCTTATGCTTGTTGGAAGCATTGCTTGG GTTTTGATTTTGTTTATAATCCACTCTTAGTGGATATTCAACAGTGGGGTTATTCTGGTAACCTACAATTTAACCAT GATTTGCATTGTAATGTGCATGGACACGCACATGTAGCTTCTGCGGATGCTATTATGACGCGTTGTCTTGCAATTAA TAATGCATTTTGTCAAGATGTCAACTGGGATTTAACTTACCCTCATATAGCAAATGAGGATGAAGTCAATTCTAGCT GTAGATATTTACAACGCATGTATCTTAATGCATGTGTTGATGCTCTTAAAGTTAACGTTGTCTATGATATAGGCAAC CCTAAAGGTATAAAATGTGTTAGACGTGGAGACTTAAATTTTAGATTCTATGATAAGAATCCAATAGTACCCAATGT CAAGCAGTTTGAGTATGACTATAATCAGCACAAAGATAAGTTTGCTGATGGTCTTTGTATGTTTTGGAATTGTAATG TGGATTGTTATCCCGACAATTCCTTAGTTTGTAGGTACGACACACGAAATTTGAGTGTGTTTAACCTACCTGGTTGT AATGGTGGTAGCTTGTATGTTAACAAGCATGCATTCCACACACCTAAATTTGATCGCACTAGCTTTCGTAATTTGAA AGCTATGCCATTCTTTTTCTATGACTCATCGCCTTGCGAGACCATTCAATTGGATGGAGTTGCGCAAGACCTTGTGT CATTAGCTACGAAAGATTGTATCACAAAATGCAACATAGGCGGTGCTGTTTGTAAAAAGCACGCACAAATGTATGCA GATTTTGTGACTTCTTATAATGCAGCTGTTACTGCTGGTTTTACTTTTTGGGTTACTAATAATTTTAACCCATATAA TTTGTGGAAAAGTTTTTCAGCTCTCCAGTCTATCGACAATATTGCTTATAATATGTATAAGGGTGGTCATTATGATG CTATTGCAGGAGAAATGCCCACTATCGTAACTGGAGATAAAGTTTTTGTTATAGATCAAGGCGTAGAAAAAGCAGTT TTTTTTAATCAAACAATTCTGCCTACATCTGTAGCGTTTGAGCTGTATGCGAAGAGAAATATTCGCACACTGCCAAA CAACCGTATTTTGAAAGGTTTGGGTGTAGATGTGACTAATGGATTTGTAATTTGGGATTACACGAACCAAACACCAC TATACCGTAATACTGTTAAGGTATGTGCATATACAGACATAGAACCAAATGGCCTAATAGTGCTGTATGATGATAGA TATGGTGATTACCAGTCTTTTCTAGCTGCTGATAATGCTGTTTTAGTTTCTACACAGTGTTACAAGCGGTATTCGTA TGTAGAAATACCGTCAAACCTGCTTGTTCAGAACGGTATTCCGTTAAAAGATGGAGCGAACCTGTATGTTTATAAGC GTGTTAATGGTGCGTTTGTTACGCTACCTAACACATTAAACACACAGGGTCGCAGTTATGAAACTTTTGAACCTCGT AGTGATGTTGAGCGTGATTTTCTCGACATGTCTGAGGAGAGTTTTGTAGAAAAGTATGGTAAAGAATTAGGTCTACA GCACATACTGTATGGTGAAGTTGATAAGCCCCAATTAGGTGGTTTACACACTGTTATAGGTATGTGCAGACTTTTAC GTGCGAATAAGTTGAACGCAAAGTCTGTTACTAATTCTGATTCTGATGTCATGCAAAATTATTTTGTATTGGCAGAC AATGGTTCCTACAAGCAAGTGTGTACTGTTGTGGATTTGCTGCTTGATGATTTCTTAGAACTTCTTAGGAACATACT GAAAGAGTATGGTACTAATAAGTCTAAAGTTGTAACAGTGTCAATTGATTACCATAGCATAAATTTTATGACTTGGT TTGAAGATGGCATTATTAAAACATGTTATCCACAGCTTCAATCAGCATGGACGTGTGGTTATAATATGCCTGAACTT TATAAAGTTCAGAATTGTGTTATGGAACCTTGCAACATTCCTAATTATGGTGTTGGAATAGCGTTGCCAAGTGGTAT TATGATGAATGTGGCAAAGTATACACAACTCTGTCAATACCTTTCGAAAACAACAATGTGTGTACCGCATAATATGC GAGTAATGCATTTTGGAGCTGGAAGTGACAAAGGAGTGGCTCCAGGTAGTACTGTTCTTAAACAATGGCTCCCAGAA GGGACACTCCTTGTCGATAATGATATTGTAGACTATGTGTCTGATGCACATGTTTCTGTGCTTTCAGATTGCAATAA ATATAAGACAGAGCACAAGTTTGATCTTGTGATATCTGATATGTATACAGACAATGATTCAAAAAGAAAGCATGAAG GCGTGATAGCCAATAATGGCAATGATGACGTTTTCATATATCTCTCAAGTTTTCTTCGTAATAATTTGGCTCTAGGT GGTAGTTTTGCTGTAAAAGTGACAGAGACAAGTTGGCACGAAGTTTTATATGACATTGCACAGGATTGTGCATGGTG GACAATGTTTTGTACAGCAGTGAATGCCTCTTCTTCAGAAGCATTCTTGGTTGGTGTTAATTATTTGGGTGCAAGTG AAAAGGTTAAGGTTAGTGGAAAAACGCTGCACGCAAATTATATATTTTGGAGGAATTGTAATTATTTACAAACCTCT GCTTATAGTATATTTGACGTTGCTAAGTTTGATTTGAGATTGAAAGCAACACCAGTTGTTAATTTGAAAACTGAACA AAAGACAGACTTAGTCTTTAATTTAATTAAGTGTGGTAAGTTACTGGTAAGAGATGTTGGTAACACCTCTTTTACTA GTGACTCTTTTGTGTGTACTATGTAGTGCTGCTTTGTATGACAGTAGTTCTTACGTTTACTACTACCAAAGTGCCTT TAGACCACCTAATGGTTGGCATTTACACGGGGGTGCTTATGCGGTAGTTAATATTTCTAGCGAATCTAATAATGCAG GCTCTTCACCTGGGTGTATTGTTGGTACTATTCATGGTGGTCGTGTTGTTAATGCTTCTTCTATAGCTATGACGGCA CCGTCATCAGGTATGGCTTGGTCTAGCAGTCAGTTTTGTACTGCACACTGTAACTTTTCAGATACTACAGTGTTTGT TACACATTGTTATAAATATGATGGGTGTCCTATAACTGGCATGCTTCAAAAGAATTTTTTACGTGTTTCTGCTATGA AAAATGGCCAGCTTTTCTATAATTTAACAGTTAGTGTAGCTAAGTACCCTACTTTTAAATCATTTCAGTGTGTTAAT AATTTAACATCCGTATATTTAAATGGTGATCTTGTTTACACCTCTAATGAGACCACAGATGTTACATCTGCAGGTGT TTATTTTAAAGCTGGTGGACCTATAACTTATAAAGTTATGAGAGAAGTTAAAGCCCTGGCTTATTTTGTTAATGGTA CTGCACAAGATGTTATTTTGTGTGATGGATCACCTAGAGGCTTGTTAGCATGCCAGTATAATACTGGCAATTTTTCA GATGGCTTTTATCCTTTTATTAATAGTAGTTTAGTTAAGCAGAAGTTTATTGTCTATCGTGAAAATAGTGTTAATAC TACTTTTACGTTACACAATTTCACTTTTCATAATGAGACTGGCGCCAACCCTAATCCTAGTGGTGTTCAGAATATTC AAACTTACCAAACACAAACAGCTCAGAGTGGTTATTATAATTTTAATTTTTCCTTTCTGAGTAGTTTTGTTTATAAG GAGTCTAATTTTATGTATGGATCTTATCACCCAAGTTGTAATTTTAGACTAGAAACTATTAATAATGGCTTGTGGTT TAATTCACTTTCAGTTTCAATTGCTTACGGTCCTCTTCAAGGTGGTTGCAAGCAATCTGTCTTTAGTGGTAGAGCAA CTTGTTGTTATGCTTATTCATATGGAGGTCCTTCGCTGTGTAAAGGTGTTTATTCAGGTGAGTTAGATCTTAATTTT GAATGTGGACTGTTAGTTTATGTTACTAAGAGCGGTGGCTCTCGTATACAAACAGCCACTGAACCGCCAGTTATAAC TCGACACAATTATAATAATATTACTTTAAATACTTGTGTTGATTATAATATATATGGCAGAACTGGCCAAGGTTTTA TTACTAATGTAACCGACTCAGCTGTTAGTTATAATTATCTAGCAGACGCAGGTTTGGCTATTTTAGATACATCTGGT TCCATAGACATCTTTGTTGTACAAGGTGAATATGGTCTTACTTATTATAAGGTTAACCCTTGCGAAGATGTCAACCA GCAGTTTGTAGTTTCTGGTGGTAAATTAGTAGGTATTCTTACTTCACGTAATGAGACTGGTTCTCAGCTTCTTGAGA ACCAGTTTTACATTAAAATCACTAATGGAACACGTCGTTTTAGACGTTCTATTACTGAAAATGTTGCAAATTGCCCT TATGTTAGTTATGGTAAGTTTTGTATAAAACCTGATGGTTCAATTGCCACAATAGTACCAAAACAATTGGAACAGTT TGTGGCACCTTTACTTAATGTTACTGAAAATGTGCTCATACCTAACAGTTTTAATTTAACTGTTACAGATGAGTACA TACAAACGCGTATGGATAAGGTCCAAATTAATTGTCTGCAGTATGTTTGTGGCAATTCTCTGGATTGTAGAGATTTG TTTCAACAATATGGGCCTGTTTGTGACAACATATTGTCTGTAGTAAATAGTATTGGTCAAAAAGAAGATATGGAACT TTTGAATTTCTATTCTTCTACTAAACCGGCTGGTTTTAATACACCATTTCTTAGTAATGTTAGCACTGGTGAGTTTA ATATTTCTCTTCTGTTAACAACTCCTAGTAGTCCTAGAAGGCGTTCTTTTATTGAAGACCTTCTATTTACAAGCGTT GAATCTGTTGGATTACCAACAGATGACGCATACAAAAATTGCACTGCAGGACCTTTAGGTTTTCTTAAGGACCTTGC GTGTGCTCGTGAATATAATGGTTTGCTTGTGTTGCCTCCCATTATAACAGCAGAAATGCAAATTTTGTATACTAGTT CTCTAGTAGCTTCTATGGCTTTTGGTGGTATTACTGCAGCTGGTGCTATACCTTTTGCCACACAACTGCAGGCTAGA ATTAATCACTTGGGTATTACCCAGTCACTTTTGTTGAAGAATCAAGAAAAAATTGCTGCTTCCTTTAATAAGGCCAT TGGTCGTATGCAGGAAGGTTTTAGAAGTACATCTCTAGCATTACAACAAATTCAAGATGTTGTTAATAAGCAGAGTG CTATTCTTACTGAGACTATGGCATCACTTAATAAAAATTTTGGTGCTATTTCTTCTATGATTCAAGAAATCTACCAG CAACTTGACGCCATACAAGCAAATGCTCAAGTGGATCGTCTTATAACTGGTAGATTGTCATCACTTTCTGTTTTAGC ATCTGCTAAGCAGGCGGAGCATATTAGAGTGTCACAACAGCGTGAGTTAGCTACTCAGAAAATTAATGAGTGTGTTA AGTCACAGTCTATTAGGTACTCCTTTTGTGGTAATGGACGACATGTTCTAACCATACCGCAAAATGCACCTAATGGT ATAGTGTTTATACACTTTTCTTATACTCCAGATAGTTTTGTTAATGTTACTGCAATAGTGGGTTTTTGTGTAAAGCC AGCTAATGCTAGTCAGTATGCAATAGTACCCGCTAATGGTAGGGGTATTTTTATACAAGTTAATGGTAGTTACTACA TCACAGCACGAGATATGTATATGCCAAGAGCTATTACTGCAGGAGATATAGTTACGCTTACTTCTTGTCAAGCAAAT TATGTAAGTGTAAATAAGACCGTCATTACTACATTCGTAGACAATGATGATTTTGATTTTAATGACGAATTGTCAAA ATGGTGGAATGACACTAAGCATGAGCTACCAGACTTTGACAAATTCAATTACACAGTACCTATACTTGACATTGATA GTGAAATTGATCGTATTCAAGGCGTTATACAGGGTCTTAATGACTCTTTAATAGACCTTGAAAAACTTTCAATACTC AAAACTTATATTAAGTGGCCTTGGTATGTGTGGTTAGCCATAGCTTTTGCCACTATTATCTTCATCTTAATACTAGG ATGGGTTTTCTTCATGACTGGATGTTGTGGTTGTTGTTGTGGATGCTTTGGCATTATGCCTCTAATGAGTAAGTGTG GTAAGAAATCTTCTTATTACACGACTTTTGATAACGATGTGGTAACTTAACAATACAGACCTAAAAAGTCTGTTTAA TGATTCAAAGTCCCACGTCCTTCCTAATAGTATTAATTTTTCTTTGGTGTAAACTTGTACTAAGTTGTTTTAGAGAG TTTATTATAGCGCTCCAACAACTAATACAAGTTTTACTCCAAATTATCAATAGTAACTTACAGCCTAGACTGACCCT TTGTCACAGTCTAGACTAATGTTAAACTTAGAAGCAATTATTGAAACTGGTGAGCAAGTGATTCAAAAAATCAGTTT CAATTTACAGCATATTTCAAGTGTATTAAACACAGAAGTATTTGACCCCTTTGACTATTGTTATTACAGAGGAGGTA ATTTTTGGGAAATAGAGTCAGCTGAAGATTGTTCAGGTGATGATGAATTTATTGAATAAGTCGCTAGAGGAAAATGG AAGTTTTCTAACAGCGCTTTATATATTTGTAGGATTTTTAGCACTTTATCTTCTAGGTAGAGCACTTCAAGCATTTG TACAGGCTGCTGATGCTTGTTGTTTATTTTGGTATACATGGGTAGTAATTCCAGGAGCTAAGGGTACAGCCTTTGTA TATAAGTATACATATGGTAGAAAACTTAACAATCCGGAATTAGAAGCAGTTATTGTCAACGAGTTTCCTAAGAACGG TTGGAATAATAAAAATCCAGCAAATTTTCAAGATGTCCAACGAGACAAATTGTACTCTTGACTTTGAACAGTCAGTT GAGCTTTTTAAAGAGTATAATTTATTTATAACTGCATTCTTGTTGTTCTTAACCATAATACTTCAGTATGGCTATGC AACAAGAAGTAAGTTTATTTATATACTGAAAATGATAGTGTTATGGTGCTTTTGGCCCCTTAACATTGCAGTAGGTG TAATTTCATGTATATACCCACCAAACACAGGAGGTCTTGTCGCAGCGATAATACTTACAGTGTTTGCGTGTCTGTCT TTTGTAGGTTATTGGATCCAGAGTATTAGACTCTTTAAGCGGTGTAGGTCATGGTGGTCATTTAACCCAGAATCTAA TGCCGTAGGTTCAATACTCCTAACTAATGGTCAACAATGTAATTTTGCTATAGAGAGTGTGCCAATGGTGCTTTCTC CAATTATAAAGAATGGTGTTCTTTATTGTGAGGGTCAGTGGCTTGCTAAGTGTGAACCAGACCACTTGCCTAAAGAT ATATTTGTTTGTACACCGGATAGACGTAATATCTACCGTATGGTGCAGAAATATACTGGTGACCAAAGCGGAAATAA GAAACGGTTTGCTACGTTTGTCTATGCAAAGCAGTCAGTAGATACTGGCGAGCTAGAAAGTGTAGCAACAGGAGGGA GTAGTCTTTACACCTAAATGTGTGTGTGTAGAGAGTATTTAAAATTATTCTTTAATAGTGCCTCTATTTTAAGAGCG CATAATAGTATTATTTTTGAGGATATTAATATAAATCCTCTCTGTTTTATACTCTCTTTTCAAGAGCTATTATTTAA AAAACAGTTTTTCCACTCTTTTGTGCCAAAAACTATTGTTGTTAATGGTGTAACCTTTCAAGTAGATAATGGAAAAG TCTACTACGAAGGAAAACCAATTTTTCAGAAAGGTTGTTGTAGGTTGTGGTTGAGTTATAAAAAAGATTAAACTACC TACTACACTTATTTTTATAAGAGGCGTTTTATCTTACAAGCGCTTAATAAATACGGACGATGAAATGGCTGACTAGT TTTGTAAGGGCAGTTATTTCATGTTATAAACCCCTATTATTAACTCAATTAAGAGTATTAGATAGGTTAATCTTAGA TCATGGACCAAAACACATCTTAACGTGTGTTAGGTGCGTGATTTTGTTTCAATTAGATTTAGTTTATAGGTTGGCGT ATACGCCTACTCAATCGCTGGTATGAATAATAGTAAAGATAATCCTTTTTGCGGAGCAATAGCAAGAAAAGCGCGAA TTTATCTGAGAGAAGGATTAGATTGTGTTTACTTTCTTAACAAAGCAGGACAAGCAGAGTCTTGTCCCGCGTGTACC TCTCTAGTATTCCAGGGGAAAACTTGTGAGGAACACAAATATAATAATAATCTTTTGTCATGGCAAGCGGTAAGGCA ACTGGAAAGACAGATGCCCCAGCTCCAGTCATCAAACTAGGAGGACCAAAGCCACCTAAAGTTGGTTCTTCTGGAAA TGTATCTTGGTTTCAAGCAATAAAAGCCAAGAAGTTAAATTCACCTCCGCCTAAGTTTGAAGGTAGCGGTGTTCCTG ATAATGAAAATCTAAAACCAAGTCAGCAGCATGGATATTGGAGACGCCAAGCTAGGTTTAAGCCAGGTAAAGGTGGA AGAAAACCAGTCCCAGATGCTTGGTATTTTTACTATACTGGAACAGGACCAGCCGCTAACCTGAATTGGGGTGATAG CCAAGATGGTATAGTGTGGGTTGCTGGTAAGGGTGCTGATACTAAATTTAGATCTAATCAGGGTACTCGTGACTCTG ACAAGTTTGACCAATATCCGCTACGGTTTTCAGACGGAGGACCTGATGGTAATTTCCGTTGGGATTTCATTCCTCTG AATCGTGGCAGGAGTGGGAGATCAACAGCAGCTTCATCAGCAGCATCTAGTAGAGCACCATCACGTGAAGTTTCGCG TGGTCGCAGGAGTGGTTCTGAAGATGATCTTATTGCTCGTGCAGCAAGGATAATTCAGGATCAGCAGAAGAAGGGTT CTCGCATTACAAAGGCTAAGGCTGATGAAATGGCTCACCGCCGGTATTGCAAGCGCACTATTCCACCTAATTATAAG GTTGATCAAGTGTTTGGTCCCCGTACTAAAGGTAAGGAGGGAAATTTTGGTGATGACAAGATGAATGAGGAAGGTAT TAAGGATGGGCGCGTTACAGCAATGCTCAACCTAGTTCCTAGCAGCCATGCTTGTCTTTTCGGAAGTAGAGTGACGC CCAGACTTCAACCAGATGGGCTGCACTTGAAATTTGAATTTACTACTGTGGTCCCACGTGATGATCCGCAGTTTGAT AATTATGTAAAAATTTGTGATCAGTGTGTTGATGGTGTAGGAACACGTCCAAAAGATGATGAACCAAGACCAAAGTC ACGCTCAAGTTCAAGACCTGCAACAAGAGGAAATTCTCCAGCGCCAAGACAGCAGCGCCCTAAGAAGGAGAAAAAGC CAAAGAAGCAGGATGATGAAGTGGATAAAGCATTGACCTCAGATGAGGAGAGGAACAATGCACAGCTGGAATTTGAT GATGAACCCAAGGTAATTAACTGGGGGGATTCAGCCCTAGGAGAGAATGAACTTTGAGTAAAATTCAATAGTAAGAG TTAAGGAAGATAGGCATGTAGCTTGATTACCTACATGTCTATCGCCAGGGAAATGTCTAATTTGTCTACTTAGTAGC CTGGAAACGAACGGTAGACCCTTAGATTTTAATTTAGTTTAATTTTTAGTTTAGTTTAAGTTAGTTTAGAGTAGGTA TAAAGATGCCAGTGCCGGGGCCACGCGGAGTACGACCGAGGGTACAGCACTAGGACGCCCATTAGGGGAAGAGCTAA ATTTTAGTTTAAGTTAAGTTTAATTGGCTATGTATAGTTAAAATTTATAGGCTAGTATAGAGTTAGAGCAAAAAAAA AAAAAAAAAAAAAAAAAAA
Auxiliary protein
All coronavirus encode the auxilin gene of one group of unknown function, are not necessary to replication in vitro, still It can work in pathogenesis.IBV encodes two auxiliary genes, i.e. gene 3 and 5, they all express two kinds of auxiliary respectively Albumen 3a, 3b and 5a, 5b.
The amino acid sequence of the nucleotide sequence of gene 3 and auxilin 3a and 3b are respectively with SEQ ID in IBV MK41 NO:2-4 is provided.
SEQ ID NO:2 (3 nucleotide sequence-SEQ ID NO of gene:1 nucleotide 23831 to 24539)
CTTAACAATACAGACCTAAAAAGTCTGTTTAATGATTCAAAGTCCCACGTCCTTCCTAATAGTATTAAT TTTTCTTTGGTGTAAACTTGTACTAAGTTGTTTTAGAGAGTTTATTATAGCGCTCCAACAACTAATACAAGTTTTAC TCCAAATTATCAATAGTAACTTACAGCCTAGACTGACCCTTTGTCACAGTCTAGACTAATGTTAAACTTAGAAGCAA TTATTGAAACTGGTGAGCAAGTGATTCAAAAAATCAGTTTCAATTTACAGCATATTTCAAGTGTATTAAACACAGAA GTATTTGACCCCTTTGACTATTGTTATTACAGAGGAGGTAATTTTTGGGAAATAGAGTCAGCTGAAGATTGTTCAGG TGATGATGAATTTATTGAATAAGTCGCTAGAGGAAAATGGAAGTTTTCTAACAGCGCTTTATATATTTGTAGGATTT TTAGCACTTTATCTTCTAGGTAGAGCACTTCAAGCATTTGTACAGGCTGCTGATGCTTGTTGTTTATTTTGGTATAC ATGGGTAGTAATTCCAGGAGCTAAGGGTACAGCCTTTGTATATAAGTATACATATGGTAGAAAACTTAACAATCCGG AATTAGAAGCAGTTATTGTCAACGAGTTTCCTAAGAACGGTTGGAATAATAAAAATCCAGCAAATTTTCAAGATGTC CAACGAGACAAATTGTACTCTTGA
SEQ ID NO:3 (auxilin 3a amino acid sequences)
MIQSPTSFLIVLIFLWCKLVLSCFREFIIALQQLIQVLLQIINSNLQPRLTLCHSLD
SEQ ID NO:4 (auxilin 3b amino acid sequences)
MLNLEAIIETGEQVIQKISFNLQHISSVLNTEVFDPFDYCYYRGGNFWEIESAEDCSGDDEFIE
Replicase
In addition to structure and auxiliary gene, 2/3rds coronavirus gene group includes that rdrp gene (is located at genome 5 ' end), be expressed as two polyproteins pp1a and pp1ab, wherein pp1ab is due to caused by -1 ribosomes displacement mechanism Pp1a extends product.The protease that two kinds of polyproteins are encoded by two viroids is cut, and generally produces 16 kinds of non-structural proteins (nsp1-16)；IBV lacks nsp1, to encode nsp2-16.
Therefore, the gene 1 in IBV encodes 15 (16 in other coronavirus) non-structural proteins (nsp2-16), It is related to rna replicon and transcription.
Term " replicase protein " is herein for referring to pp1a and pp1ab polyprotein or individual nsp subunit.
Term " rdrp gene " is herein for referring to the nucleic acid sequence of coding replicase protein matter.
The general introduction of coronavirus nsp protein function is provided in table 1.
Table 1
The large protein that Nsp-3 is made of many unique domains separated by unordered connector, including ubiquitin sample domain, Acidic domain, the domain papain-like proteases (PL pro), multiple transmembrane domains and big domain.Big domain is that have with flank positioned at nsp- The spherical domain of the center web of the spiral at the end 3N.It contain ADP- ribose -1- phosphatase (ADRP) activity, and with it is various forms of ADP- ribose combines.The domain is highly conserved in coronavirus, but only finds in other several Viraceaes.
The nucleotide of nsp-3 and protein sequence are respectively with SEQ ID NO in IBV M41:5 and 6 provide.
SEQ ID NO:5 (nsp-3 nucleotide sequence-SEQ ID NO:1 nucleotide 2548-7329)
GGTAAAACTGTCACCTTTGGAGAAACTACTGTGCAAGAAATACCACCACCTGATGTTGTGTTTATTAAG GTTAGCATTGAGTGTTGTGGTGAACCATGGAATACAATCTTCAAAAAGGCTTATAAGGAGCCCATTGAAGTAGAGAC AGACCTCACAGTTGAACAATTGCTCTCTGTGGTCTATGAGAAAATGTGTGATGATCTCAAGCTGTTTCCGGAGGCTC CAGAACCACCACCATTTGAGAATGTCACACTTGTTGATAAGAATGGTAAAGATTTGGATTGCATAAAATCATGCCAT CTGATCTATCGTGATTATGAGAGCGATGATGACATCGAGGAAGAAGATGCAGAAGAATGTGACACGGATTCAGGTGA TGCTGAGGAGTGTGACACTAATTCAGAATGTGAAGAAGAAGATGAGGATACTAAAGTGTTGGCTCTTATACAAGACC CGGCAAGTAACAAATATCCTCTGCCTCTTGATGATGATTATAGCGTCTACAATGGATGTATTGTTCATAAGGACGCT CTCGATGTTGTGAATTTACCATCTGGTGAAGAAACCTTTGTTGTCAATAACTGCTTTGAAGGGGCTGTTAAAGCTCT TCCGCAGAAAGTTATTGATGTTCTAGGTGACTGGGGTGAGGCTGTTGATGCGCAAGAACAATTGTGTCAACAAGAAT CAACTCGGGTCATATCTGAGAAATCAGTTGAGGGTTTTACTGGTAGTTGTGATGCAATGGCTGAACAAGCTATTGTT GAAGAGCAGGAAATAGTACCTGTTGTTGAACAAAGTCAGGATGTAGTTGTTTTTACACCTGCAGACCTAGAAGTTGT TAAAGAAACAGCAGAAGAGGTTGATGAGTTTATTCTCATTTCTGCTGTCCCTAAAGAAGAAGTTGTGTCTCAGGAGA AAGAGGAGCCACAGGTTGAGCAAGAGCCTACCCTAGTTGTTAAAGCACAACGTGAGAAGAAGGCTAAAAAGTTCAAA GTTAAACCAGCTACATGTGAAAAACCCAAATTTTTGGAGTACAAAACATGTGTGGGTGATTTGGCTGTTGTAATTGC CAAAGCATTGGATGAGTTTAAAGAGTTCTGCATTGTAAACGCTGCAAATGAGCACATGTCGCATGGTGGTGGCGTTG CAAAGGCAATTGCAGACTTTTGTGGACCGGACTTTGTTGAATATTGCGCGGACTATGTTAAGAAACATGGTCCACAG CAAAAACTTGTCACACCTTCATTTGTTAAAGGCATTCAATGTGTGAATAATGTTGTAGGACCTCGCCATGGAGACAG CAACTTGCGTGAGAAGCTTGTTGCTGCTTACAAGAGTGTTCTTGTAGGTGGAGTGGTTAACTATGTTGTGCCAGTTC TCTCATCAGGGATTTTTGGTGTAGATTTTAAAATATCAATAGATGCTATGCGCGAAGCTTTTAAAGGTTGTGCCATA CGCGTTCTTTTATTTTCTCTGAGTCAAGAACACATCGATTATTTCGATGCAACTTGTAAGCAGAAGACAATTTATCT TACGGAGGATGGTGTTAAATACCGCTCTGTTGTTTTAAAACCTGGTGATTCTTTGGGTCAATTTGGACAGGTTTTTG CAAGAAATAAGGTAGTCTTTTCGGCTGATGATGTTGAGGATAAAGAAATCCTCTTTATACCCACAACTGACAAGACT ATTCTTGAATATTATGGTTTAGATGCGCAAAAGTATGTAACATATTTGCAAACGCTTGCGCAGAAATGGGATGTTCA ATATAGAGACAATTTTGTTATATTAGAGTGGCGTGACGGAAATTGCTGGATTAGTTCAGCAATAGTTCTCCTTCAAG CTGCTAAAATTAGATTTAAAGGTTTTCTTGCAGAAGCATGGGCTAAACTGTTGGGTGGAGATCCTACAGACTTTGTT GCCTGGTGTTATGCAAGTTGCAATGCTAAAGTAGGTGATTTTTCAGATGCTAATTGGCTTTTGGCCAATTTAGCAGA ACATTTTGACGCAGATTACACAAATGCACTTCTTAAGAAGTGTGTGTCGTGCAATTGTGGTGTTAAGAGTTATGAAC TTAGGGGTCTTGAAGCCTGTATTCAGCCAGTTCGAGCACCTAATCTTCTACATTTTAAAACGCAATATTCAAATTGC CCAACCTGTGGTGCAAGTAGTACGGATGAAGTAATAGAAGCTTCATTACCGTACTTATTGCTTTTTGCTACTGATGG TCCTGCTACAGTTGATTGTGATGAAAATGCTGTAGGGACTGTTGTTTTCATTGGCTCTACTAATAGTGGCCATTGTT ATACACAAGCCGATGGTAAGGCTTTTGACAATCTTGCTAAGGATAGAAAATTTGGAAGGAAGTCGCCTTACATTACA GCAATGTATACACGTTTTTCTCTTAGGAGTGAAAATCCCCTACTTGTTGTTGAACATAGTAAGGGTAAAGCTAAAGT AGTAAAAGAAGATGTTTCTAACCTTGCTACTAGTTCTAAAGCCAGTTTTGACGATCTTACTGACTTTGAACAGTGGT ATGATAGCAACATCTATGAGAGTCTTAAAGTGCAGGAGACACCTGATAATCTTGATGAATATGTGTCATTTACGACA AAGGAAGATTCTAAGTTGCCACTGACACTTAAAGTTAGAGGTATCAAATCAGTTGTTGACTTTAGGTCTAAGGATGG TTTTACTTATAAGTTAACACCTGATACTGATGAAAATTCAAAAACACCAGTCTACTACCCAGTCTTGGATTCTATTA GTCTTAGGGCAATATGGGTTGAAGGCAGTGCTAATTTTGTTGTTGGGCATCCAAATTATTATAGTAAGTCTCTCCGA ATTCCCACGTTTTGGGAAAATGCCGAGAGCTTTGTTAAAATGGGTTATAAAATTGATGGTGTAACTATGGGCCTTTG GCGTGCAGAACACCTTAATAAACCTAATTTGGAGAGAATTTTTAACATTGCTAAGAAAGCTATTGTTGGATCTAGTG TTGTTACTACGCAGTGTGGTAAAATACTAGTTAAAGCAGCTACATACGTTGCCGATAAAGTAGGTGATGGTGTAGTT CGCAATATTACAGATAGAATTAAGGGTCTTTGTGGATTCACACGTGGCCATTTTGAAAAGAAAATGTCCCTACAATT TCTAAAGACACTTGTGTTCTTTTTCTTTTATTTCTTAAAGGCTAGTGCTAAGAGTTTAGTTTCTAGCTATAAGATTG TGTTATGTAAGGTGGTGTTTGCTACCTTACTTATAGTGTGGTTTATATACACAAGTAATCCAGTAGTGTTTACTGGA ATACGTGTGCTAGACTTCCTATTTGAAGGTTCTTTATGTGGTCCTTATAATGACTACGGTAAAGATTCTTTTGATGT GTTACGCTATTGTGCAGGTGATTTTACTTGTCGTGTGTGTTTACATGATAGAGATTCACTTCATCTGTACAAACATG CTTATAGCGTAGAACAAATTTATAAGGATGCAGCTTCTGGCATTAACTTTAATTGGAATTGGCTTTATTTGGTCTTT CTAATATTATTTGTTAAGCCAGTGGCAGGTTTTGTTATTATTTGTTATTGTGTTAAGTATTTGGTATTGAGTTCAAC TGTGTTGCAAACTGGTGTAGGTTTTCTAGATTGGTTTGTAAAAACAGTTTTTACCCATTTTAATTTTATGGGAGCGG GATTTTATTTCTGGCTCTTTTACAAGATATACGTACAAGTGCATCATATATTGTACTGTAAGGATGTAACATGTGAA GTGTGCAAGAGAGTTGCACGCAGCAACAGGCAAGAGGTTAGCGTTGTAGTTGGTGGACGCAAGCAAATAGTGCATGT TTACACTAATTCTGGCTATAACTTTTGTAAGAGACATAATTGGTATTGTAGAAATTGTGATGATTATGGTCACCAAA ATACATTTATGTCCCCTGAAGTTGCTGGCGAGCTTTCTGAAAAGCTTAAGCGCCATGTTAAACCTACAGCATATGCT TACCACGTTGTGTATGAGGCATGCGTGGTTGATGATTTTGTTAATTTAAAATATAAGGCTGCAATTCCTGGTAAGGA TAATGCATCTTCTGCTGTTAAGTGTTTCAGTGTTACAGATTTTTTAAAGAAAGCTGTTTTTCTTAAGGAGGCATTGA AATGTGAACAAATATCTAATGATGGTTTTATAGTGTGTAATACACAGAGTGCGCATGCACTAGAGGAAGCAAAGAAT GCAGCCGTCTATTATGCGCAATATCTGTGTAAGCCAATACTTATACTTGACCAGGCACTTTATGAGCAATTAATAGT AGAGCCTGTGTCTAAGAGTGTTATAGATAAAGTGTGTAGCATTTTGTCTAATATAATATCTGTAGATACTGCAGCTT TAAATTATAAGGCAGGCACACTTCGTGATGCTCTGCTTTCTATTACTAAAGACGAAGAAGCCGTAGATATGGCTATC TTCTGCCACAATCATGAAGTGGAATACACTGGTGACGGTTTTACTAATGTGATACCGTCATATGGTATGGACACTGA TAAGTTGACACCTCGTGATAGAGGGTTTTTGATAAATGCAGATGCTTCTATTGCTAATTTAAGAGTCAAAAATGCTC CTCCGGTAGTATGGAAGTTTTCTGATCTTATTAAATTGTCTGACAGTTGCCTTAAATATTTAATTTCAGCTACTGTC AAGTCAGGAGGTCGTTTCTTTATAACAAAGTCTGGTGCTAAACAAGTTATTTCTTGTCATACCCAGAAACTGTTGGT AGAGAAAAAGGCAGGT
SEQ ID NO:6 (nsp-3 amino acid sequences)
GKTVTFGETTVQEIPPPDVVFIKVSIECCGEPWNTIFKKAYKEPIEVETDLTVEQLLSVVYEKMCDDLK LFPEAPEPPPFENVTLVDKNGKDLDCIKSCHLIYRDYESDDDIEEEDAEECDTDSGDAEECDTNSECEEEDEDTKVL ALIQDPASNKYPLPLDDDYSVYNGCIVHKDALDVVNLPSGEETFVVNNCFEGAVKALPQKVIDVLGDWGEAVDAQEQ LCQQESTRVISEKSVEGFTGSCDAMAEQAIVEEQEIVPVVEQSQDVVVFTPADLEVVKETAEEVDEFILISAVPKEE VVSQEKEEPQVEQEPTLVVKAQREKKAKKFKVKPATCEKPKFLEYKTCVGDLAVVIAKALDEFKEFCIVNAANEHMS HGGGVAKAIADFCGPDFVEYCADYVKKHGPQQKLVTPSFVKGIQCVNNVVGPRHGDSNLREKLVAAYKSVLVGGVVN YVVPVLSSGIFGVDFKISIDAMREAFKGCAIRVLLFSLSQEHIDYFDATCKQKTIYLTEDGVKYRSVVLKPGDSLGQ FGQVFARNKVVFSADDVEDKEILFIPTTDKTILEYYGLDAQKYVTYLQTLAQKWDVQYRDNFVILEWRDGNCWISSA IVLLQAAKIRFKGFLAEAWAKLLGGDPTDFVAWCYASCNAKVGDFSDANWLLANLAEHFDADYTNALLKKCVSCNCG VKSYELRGLEACIQPVRAPNLLHFKTQYSNCPTCGASSTDEVIEASLPYLLLFATDGPATVDCDENAVGTVVFIGST NSGHCYTQADGKAFDNLAKDRKFGRKSPYITAMYTRFSLRSENPLLVVEHSKGKAKVVKEDVSNLATSSKASFDDLT DFEQWYDSNIYESLKVQETPDNLDEYVSFTTKEDSKLPLTLKVRGIKSVVDFRSKDGFTYKLTPDTDENSKTPVYYP VLDSISLRAIWVEGSANFVVGHPNYYSKSLRIPTFWENAESFVKMGYKIDGVTMGLWRAEHLNKPNLERIFNIAKKA IVGSSVVTTQCGKILVKAATYVADKVGDGVVRNITDRIKGLCGFTRGHFEKKMSLQFLKTLVFFFFYFLKASAKSLV SSYKIVLCKVVFATLLIVWFIYTSNPVVFTGIRVLDFLFEGSLCGPYNDYGKDSFDVLRYCAGDFTCRVCLHDRDSL HLYKHAYSVEQIYKDAASGINFNWNWLYLVFLILFVKPVAGFVIICYCVKYLVLSSTVLQTGVGFLDWFVKTVFTHF NFMGAGFYFWLFYKIYVQVHHILYCKDVTCEVCKRVARSNRQEVSVVVGGRKQIVHVYTNSGYNFCKRHNWYCRNCD DYGHQNTFMSPEVAGELSEKLKRHVKPTAYAYHVVYEACVVDDFVNLKYKAAIPGKDNASSAVKCFSVTDFLKKAVF LKEALKCEQISNDGFIVCNTQSAHALEEAKNAAVYYAQYLCKPILILDQALYEQLIVEPVSKSVIDKVCSILSNIIS VDTAALNYKAGTLRDALLSITKDEEAVDMAIFCHNHEVEYTGDGFTNVIPSYGMDTDKLTPRDRGFLINADASIANL RVKNAPPVVWKFSDLIKLSDSCLKYLISATVKSGGRFFITKSGAKQVISCHTQKLLVEKKAG
That reduces is pathogenic
Attenuation coronavirus living of the invention includes at least one mutation, and the mutation is so that virus is preced with corresponding wild type Shape virus is compared pathogenic with what is reduced.
As used herein, term " attenuation ", which refers to, shows the pathogenic of the reduction and can be classified as non-toxic Virus.Attenuated virus living is a kind of duplicating virus of reduction, still is able to stimulation immune response and generates immunity But do not cause actual disease.
Term " pathogenic " is used according to its normal meaning herein, refers to that virus causes the possibility of disease in subject Property.In general, the pathogenic of coronavirus is determined by measurement disease related symptom, such as wheezing, hair click and tracheae are fine Knitting is reduced.
Term is " reduction pathogenic " for describing the pathogenic of compared with corresponding wild type coronavirus coronavirus Level is reduced, mitigates or is reduced.
In one embodiment, coronavirus of the invention with it is derivative it parent M41-CK virus or compare coronal disease Poison is pathogenic compared to what is reduced.Control coronavirus can be with known pathogenic coronavirus.
The pathogenic of method assessment coronavirus well known in the art can be used.In general, by measurement by virus attack Subject (such as chicken) clinical symptoms it is pathogenic to assess.
As explanation, chicken is attacked when can be seeded in 8-24 days ages by nose or eye.It can be with 3-10 after infection It assesses clinical symptoms relevant to IBV infection.Usually assess the pathogenic clinical condition to determine coronavirus (such as IBV) Shape includes wheezing, beating cough, wheezing, hair click, forces down (depression), gauffer feather and tracheae ciliary activity and loses.
Compared with corresponding wild type coronavirus, attenuation coronavirus of the invention can cause reduced clinical symptoms It is horizontal.
For example, the click number that attenuation coronavirus can cause every birds per minute, is by wild-type virus Caused click number less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, be less than 30%, less than 20% or less than 10%.
Attenuation coronavirus according to the present invention birds numbers can be less than in the fowl group of infection wild-type virus 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20% or less than 10% in cause wheezing.
Attenuation coronavirus according to the present invention can cause tracheal cilia active, be to be uninfected by tracheal cilia in birds At least 60%, at least 70%, at least 80%, at least 90% or at least the 95% of activity level.
Attenuation coronavirus according to the present invention can be caused with lower level such as institute in table 2 than wild type coronavirus The clinical symptoms of definition.
Table 2：IBV severity limit based on clinical sign：
Table 2
Attenuation coronavirus of the invention can be in ovum with non-pathogenic horizontal duplication.
When being applied to the vaccine of chicken embryo in exploitation ovum, it has to be noted that two o'clock：Influence and epidemic disease of the maternal antibody to vaccine Influence of the seedling to embryo.Known maternal antibody interferes active immunity.For example, the vaccine with slight strain is being applied to parent Not eliciting protective antibody level when the broiler chicken of antibody, because these strains are neutralized by maternal antibody library.
Therefore, virion must enough effectively, to ensure it not derived from parent for disease in duplication and breeding The antibody of poison neutralizes.Antibody derived from parent is the limited library of potent antibodies, is reduced as chicken grows up, and by this method Virus is neutralized not equal to foundation to embryo/chicken permanent immunity.In order to form the permanent immunity power to virus, embryo and hatching Chicken necessarily is formed protective immune response appropriate, this is different from the effect of maternal antibody.
In order to can be used for the inoculation of ovum intradermal vaccine, virus also must not be to lead to its pathogenic to embryo horizontal duplication and numerous It grows.
The pathogenic coronavirus that may mean that attenuation reduced for embryo and corresponding wild type control are coronal Virus is less compared to causing hatching rate to reduce.Therefore, in the context of the present invention, term " non-pathogenic to embryo " can refer to " reduced hatching rate is not caused " when compared with compareing coronavirus.
The suitable attenuation coronavirus of methods known in the art identification can be used.For example, can carry out having or There is no the comparison attacks in the case where antibody derived from parent in the ovum of embryo after vaccine inoculation to test (that is, wherein laying hen Or not yet it is directed to IBV vaccine inoculation).
If the coronavirus of attenuation will not be survived (i.e. after will not hatching or hatching with excessively high level breeding, embryo Virus is pathogenic to embryo).The virus caused a disease to embryo can kill embryo.
If the coronavirus of attenuation shows that the reduction of virus replication and breeding is too big, virus will resist derived from parent Body neutralizes.Therefore, with IBV to the subsequent attack of chicken will lead to chicken under fire clinical symptoms (such as wheezing, hair click, Ciliary activity lose) formation and seizure of disease because it will cannot be formed for virus effective immunity.
Variant
As used herein, term " variant " is synonymous with " mutant ", and refers to different with corresponding wild-type sequence Nucleic acid or amino acid sequence.
Variant/mutant sequence can be naturally-produced, or can be with manual creation (such as passing through direct mutagenesis).Mutant There can be at least 70%, 80%, 90%, 95%, 98% or 99% sequence identity with the corresponding portion of wild-type sequence. Mutant can have 20,10,5,4,3,2 or 1 mutation in the corresponding portion of wild-type sequence.
Term " wild type " is for referring to identical as natural gene or protein (i.e. viral gene or protein) respectively Nucleotide or amino acid sequence gene or protein.
Identity can relatively use eye, or more generally carry out by means of being easy obtainable sequence comparison program.These The computer program of commercialization can calculate the % identity between two or more sequences.It is suitable for carrying out such comparison Computer program is GCG Wisconsin Bestfit packet (University of Wisconsin, U.S.A.；Devereux et al.,1984,Nucleic Acids Research 12:387).It can carry out the example packet of the other software of sequence comparison Include but be not limited to BLAST packet (referring to Ausubel et al., 1999-the 18 chapters as above), FASTA (Atschul et al., 1990, J.Mol.Biol., 403-410) and GENEWORKS compare suite of tools ClustalX (referring to Larkin et al. (2007)Clustal W and Clustal X version 2.0.Bioinformatics,23:2947-2948)。BLAST It can be used in offline and on-line search (being same as above referring to Ausubel etc., 1999,7-58 to 7-60 pages) with FASTA.But it is right In certain applications, it is preferable to use GCG Bestfit program.The new tool of referred to as BLAST 2Sequences can also be used for comparing egg White matter and nucleotide sequence are (referring to FEMS Microbiol Lett 1,999 174 (2):247-50；FEMS Microbiol Lett 1999 177(1):187-8 and tatiana@ncbi.nlm.nih.gov).
Sequence can have one or more missings, insertion or the substitution of amino acid residue, generates silencing and changes and produce The equivalent molecule of raw function.As long as retentive activity, can polarity based on residue, charge, solubility, hydrophobicity, hydrophily and/ Or the similitude of amphipathic characteristic carries out intentional amino acid substitution.For example, negatively charged amino acid include aspartic acid and Glutamic acid；Positively charged amino acid includes lysine and arginine；Contain uncharged pole with similar hydrophilicity score The amino acid of property headgroup includes leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, silk ammonia Acid, threonine, phenylalanine and tyrosine.
Conservative substitution can be carried out, such as according to following table.Amino acid in secondary series in same block and preferably in third The amino acid in mutually colleague in column can replace each other：
Ammayappan et al(Arch Virol(2009)154:495-499) report responsible IBV plants of Arkansas The identification of the sequence variation of DPI attenuation.The multiple passage of virus of the research in embryonated egg, identifies after about 100 times a variety of 17 kinds of amino acid variation in IBV albumen.Whether do not study attenuated virus (Ark DPI 101) can be in ovum for disease It is replicated in the presence of antibody derived from the parent of poison, and is not pathogenic to embryo.It is by SPF embryonated egg in view of the virus Repeatedly passage generates, for method as classical IBV vaccine, it is possible to which such virus is pathogenic to embryo.If female The similar serotype vaccine inoculation of chicken, then virus may also be sensitive to antibody derived from parent.
Coronavirus of the invention includes mutation and/or auxiliary protein 3a and/or 3b in nonstructural proteins nsp-3 Missing, the mutation cause virus with wild type coronavirus compared with have reduction it is pathogenic.
Mutation in nsp-3 may include prominent in adenosine diphosphate-region ribose -1 '-phosphatase (ADRP) of nsp-3 Become.The area ADRP of nsp-3 may include SEQ ID NO:6 residue 332 to 491 or by SEQ ID NO:6 residue 332 to 491 compositions are (for example, by SEQ ID NO:5 nucleotide 994 to 1473 encodes).Therefore, with SEQ ID NO:The 332 to 491 of 6 Amino acid shown in position is compared, and the mutation in nsp-3 may include one or more amino acid mutations.Mutation in nsp-3 with SEQ ID NO:6 position 332 to 491,350 to 470,350 to 450,350 to 425,350 to 400,350 to 380,360 to Amino acid shown in 380 or 370 to 380, which is compared, may include one or more amino acid mutations.Mutation and SEQ in nsp-3 ID NO:Amino acid shown in 6 position 370-380, which is compared, may include one or more amino acid mutations.
Mutation in nsp-3 may include one or more amino acid mutations selected from the group below：
a)SEQ ID NO:373 Asn (N) to Ala (A) in 6；With
b)SEQ ID NO:379 Gly (G) to Ser (S) in 6.
Mutation in nsp-3 may include one or more nucleotide and replace, and it is prominent to encode amino acid as described herein Become.
Mutation in nsp-3 may include one or more nucleotide selected from the group below and replace：
A) with SEQ ID NO:Sequence shown in 5 is compared at A to G and nucleotide position 1117 at nucleotide position 1116 A to C；With
B) with SEQ ID NO:Sequence shown in 5 is compared to the G to A at nucleotide position 1138.
As used herein, term " substitution " is mutated synonymous with term, and means to identify that the nucleotide at position is different from The nucleotide of wild-type nucleotide sequences.
Mutation in nsp-3 may include SEQ ID NO:373 amino acid mutation Asn (N) to Ala (A) in 3.
Mutation in nsp-3 may include SEQ ID NO:379 amino acid mutation Gly (G) to Ser (S) in 3.
Mutation in nsp-3 may include SEQ ID NO:In 3 373 amino acid mutation Asn (N) to Ala (A) and 379 Gly (G) to Ser (S).
The coronavirus of attenuation can also be defined on nucleotide level.
For example, the nucleotide sequence of attenuation coronavirus of the invention may include it is selected from the group below one or more prominent Become：
A) with SEQ ID NO:Sequence shown in 5 is compared at A to G and nucleotide position 1117 at nucleotide position 1116 A to C；With
B) with SEQ ID NO:Sequence shown in 5 is compared to the G to A at nucleotide position 1138.
Mutation and SEQ ID NO in nsp-3:Sequence shown in 5, which is compared, may include dashing forward at nucleotide position 1116 Become the mutation A to C at A to G and nucleotide position 1117.
Mutation and SEQ ID NO in nsp-3:Sequence shown in 5, which is compared, may include dashing forward at nucleotide position 1138 Become G to A.
Mutation and SEQ ID NO in nsp-3:Sequence shown in 5, which is compared, may include dashing forward at nucleotide position 1116 The G to A at A to C and nucleotide position 1138 at change A to G, nucleotide position 1117.
The attenuation coronavirus of work of the invention may include the missing of auxilin 3a and/or 3b.The mutation can lead to The partially or completely missing of gene 3 is crossed to realize.Therefore, in one aspect of the invention, lack auxilin 3a whole or Part.According to another aspect of the present invention, all or part of auxilin 3b is lacked.In another aspect of this invention, it lacks The all or part of both auxilin 3a and 3b.In one aspect of the invention, auxilin 3a is lacked.It is according to the present invention On the other hand, auxilin 3b is lacked.In another aspect of this invention, both auxilin 3a and 3b are lacked.
The missing of auxilin 3a and 3b may include from SEQ ID NO:The about nucleotide position of 3 sequence of gene shown in 2 Set the missing of 37 to about 384.The missing of auxilin 3a and 3b may include from SEQ ID NO:3 sequence of gene shown in 2 The missing of nucleotide position 37 to 384
The missing of auxilin 3a and 3b may include from SEQ ID NO:The about nucleosides of IBV M41-CK sequence shown in 1 The missing of sour position 23867 to about 24214.The missing of auxilin 3a and 3b may include from SEQ ID NO:IBV shown in 1 The missing of the nucleotide position 23867 to 24214 of M41-CK sequence.
Nucleotide sequence may include any combination of coding mutation listed above.
In one embodiment, coronavirus of the invention may include mutation in nonstructural proteins nsp-3 and The missing of auxiliary protein 3a and/or 3b.
In another embodiment, coronavirus of the invention may include mutation in the region ADRP of nsp-3 and The missing of auxilin 3a and 3b.
For example, the nucleotide sequence of attenuation coronavirus of the invention may include following mutation：
A) with SEQ ID NO:Sequence shown in 5 is compared at A to G and nucleotide position 1117 at nucleotide position 1116 A to C；And/or
B) with SEQ ID NO:Sequence shown in 5 is compared to the G to A at nucleotide position 1138；With
C) from SEQ ID NO:The missing of the nucleotide position 37 to 384 of 3 sequence of gene shown in 2.
Nucleotide sequence can not include take corresponding with the C12008T substitution of Ammayappan et al. report (as above) Generation.
Nucleotide sequence can be natural, synthesis or recombination.It can be double-strand or single-stranded, it can be DNA Or RNA or combinations thereof.For example, it can be cDNA, PCR product, genome sequence or mRNA.
Codon optimization can be carried out to nucleotide sequence to generate in selected host/host cell.
It can be separation, or a part as plasmid, virus or host cell.
Plasmid
Plasmid is the extrachromosomal dna molecule separated with chromosomal DNA, can be replicated independently of chromosomal DNA.They It is usually cyclic annular and double-strand.
Plasmid can be used or carrier (such as they are sometimes known) expresses protein in host cell.For example, can use It is capable of the plasmid transfection bacterial host cell of encoding specific proteins to express the protein.The term further includes Yeast Artificial's dye Colour solid and bacterial artificial chromosome, they can accommodate the longer part DNA.
Plasmid of the invention includes the nucleotide sequence that can encode the limited area of attenuation coronavirus.It can also be wrapped Containing one or more other coronavirus nucleotide sequences, or one or more other coronavirus proteins such as S can be encoded The nucleotide sequence of gene.
Plasmid can also include resistance markers, such as the Huang of the guanine from Escherichia coli (Escherichia coli) Purine phosphoribosyl transferase gene (gpt) assigns in the presence of xanthine and hypoxanthine and resisting to mycophenolic acid (MPA) Property, and controlled by vaccinia virus P7.5 early late promoter.
Recombinant vaccine virus
The invention further relates to vaccinia virus recombinant (rVV), and it includes variant genes as herein defined.
The preparation of the reverse genetics system based on vaccinia virus can be used in vaccinia virus recombinant (rVV).
In this respect, the present invention also provides pass through the following method for generating virion：
(i) plasmid transfection as described in prosthomere is entered in host cell；
(ii) recombinant virus infection host cell is used, the recombinant virus includes coronal with nsp-3 gene or gene 3 The genome of Strain；
(iii) allow that homologous recombination occurs between the corresponding sequence in gene order and recombinant virus genomes in plasmid To generate modified gene；
(iv) selection includes the recombinant virus of modified gene.
Term " modified gene " refers to nsp-3 gene or gene 3, and it includes in conjunction with as described in first aspect present invention Mutation.Specifically, the term refer to from wild type gene but include so that its is encoded misfolded proteins as herein defined, or The gene of (in the case where gene 3) not nucleotide sequence of some or all of coding protein 3a and 3b
Recombination can be related to all or part of modified gene.Recombination can be related to encoding the nucleosides of amino acid mutation Acid sequence replaces comprising nucleotide as defined above.
The genome of coronavirus strain can lack the protein portion of the part provided corresponding to plasmid, therefore by inserting The nucleotide sequence for entering plasmid offer forms the protein of modification.
Recombinant virus is suitable for carrying out the virus of homologous recombination between its genome and plasmid.Vaccinia virus is especially suitable It closes, because of the conventional genome sequence for being inserted into and lacking vaccinia virus using homologous recombination.
Optionally, the above method includes the following steps：
(v) recombination coronavirus of the recycling comprising modified gene from the DNA of the recombinant virus from step (iv).
Method for recycling recombination coronavirus (such as recombination IBV) is known in the art (referring to Britton (2005) referring to page 24；And PCT/GB2010/001293).
For example, in the DNA insertion plasmid of the recombinant virus of step (iv) and transfection expression cytoplasm can will be used for The cell of t7 rna polymerase.For example, the preparatory infection cell of fowlpox virus of expression t7 rna polymerase can be used.It then can example The separation recombination coronavirus such as from growth medium.
When plasmid is inserted into vaccinia virus gene group, unstable intermediate is formed.It is, for example, possible to use on plasmid Resistance markers selection includes the recombinant of plasmid.
It is then possible to contain modified gene for example, by PCR and sequence verification positive recombinants.
A large amount of stostes of recombinant virus can be cultivated, the recombinant virus includes modified gene (such as recombinant vaccinia Virus, (rVV), and DNA is extracted to carry out step (v)).
Suitable reverse genetics system is (Casais et al (2001) J.Virol 75 known in the art: 12359-12369；Casais et al(2003)J.Virol.77:9084-9089；Britton et al(2005) J.Virological Methods 123:203-211；Armesto et al(2008)Methods in Molecular Biology 454:255-273)。
Cell
Coronavirus can be used and carry out infection cell.
For example, Coronavirus particles can be harvested from supernatant by methods known in the art, and optionally purify.
Cell can be used to generate Coronavirus particles.
Therefore, the present invention also provides the method for being used to prepare coronavirus, this approach includes the following steps：
(i) with coronavirus infection cell according to the present invention；
(ii) virus is allowed to replicate in cell；With
(iii) progeny virus is harvested.
The present invention also provides the cells for using reverse genetics system that can generate coronavirus according to the present invention.Example Such as, cell may include recombinant virus genomes, and it includes the nucleotide sequences that can encode modified gene of the invention.
Cell can generate the recombinant recombinant virus (such as vaccinia virus) containing mutation.
Alternatively, cell can generate recombination coronavirus by reverse genetics system.Cell can be expressed or be passed through Inducing expression T7 polymerase is to save recombinant virus particle.
Vaccine
Coronavirus can be used to produce vaccine.Vaccine can be the attenuated live form of coronavirus of the invention, and And pharmaceutical acceptable carrier can be further included.As defined herein, being suitable for the invention " pharmaceutical acceptable carrier " is Well known to a person skilled in the art.Examples of such carriers includes but is not limited to water, salt water, buffered saline, phosphate buffer, alcohol/water Solution, lotion or suspension.Other conventional use of diluents and excipient can be added according to routine techniques.Examples of such carriers can To include the organic ester of ethyl alcohol, polyalcohol and its suitable mixture, vegetable oil and injectable.Also buffer and pH can be used Regulator.Buffer includes but is not limited to the salt prepared by organic acid or alkali.Representative buffer includes but is not limited to organic acid Salt, such as the salt of citric acid, such as citrate, ascorbic acid, gluconic acid, histidine-Hel, carbonic acid, tartaric acid, succinic acid, second Acid or phthalic acid, Tris, trimethylamine hydrochloride or phosphate buffer.Parenteral carrier may include sodium chloride solution, woods Grignard dextrose, dextrose, trehalose, sucrose and sodium chloride, Lactated Ringer'S Solution or fixed oil.Intravenous vehicles can be with Including liquid and nutritional supplement, electrolyte replenisher, such as based on those of woods grignard dextrose etc..In pharmaceutical carrier Preservative and other additives, such as antimicrobial, antioxidant, chelating agent (such as EDTA), inertia can be provided Gas etc..The present invention is not limited by carrier selection.By with the upper of pH isotonicty appropriate, stability and other general characteristics It states component and prepares these pharmaceutically acceptable compositions within the skill of the art.See, e.g., such as Remington: The Science and Practice of Pharmacy, the 20th edition, Lippincott Williams&Wilkins, pub！.,2000；With The Handbook of Pharmaceutical Excipients, the 4th addendum, R.C.Rowe et Al is compiled, APhA Publications, 2003 textbook.
Vaccine of the invention will be applied with " therapeutically effective amount ", and the therapeutically effective amount, which refers to work as, is applied to subject or patient When be enough to realize the amount of the active constituent (for example, reagent according to the present invention) of beneficial or desired result.It can be primary or more Effective quantity is applied in secondary application, application or dosage.Those of ordinary skill in the art can readily determine that according to the present invention group Close the therapeutically effective amount of object.In the context of the present invention, " therapeutically effective amount " is to be enough to realize the production of beneficial or desired result The amount of the objective measurement variation of raw one or more parameters relevant to the infective bronchitis patient's condition.It can be one or many Effective quantity is applied in application.For the purpose of the present invention, a effective amount of drug, compound or pharmaceutical composition are to be enough to reduce biography The amount of metachromia bronchitis disease incidence.As used herein, term " therapeutic agent " covers the entire of the treatment of disease, the patient's condition or illness Range." treatment " agent of the invention can by precaution or it is preventative in a manner of work, including those incorporations are designed as targeting Those of program of risky animal (pharmacogenetics) can be accredited as；Or it is acted as in a manner of improvement or therapeutic properties With；Or it can work to slow down the speed or degree of at least one symptom of the disease for the treatment of or illness progress.
The invention further relates to the method for preparing such vaccine, this method includes being defined with containing such as first aspect present invention Modified albumen infestation with virus particles cell, such as the step of Vero cell.
Method of vaccination
Coronavirus of the invention can be used to treat and/or prevent disease.
" treatment " refers to vaccine administration in the subject with present illness, with mitigation, reduction or improves and disease phase At least one symptom of pass and/or the progress for slowing down, reducing or blocking disease.
" prevention " refers to vaccine administration in not yet catching and/or do not show the subjects of any disease symptoms, with The reason of prevention or damage disease (such as infection) or the formation for reducing or preventing at least one symptom relevant to disease.
Disease can be any disease as caused by coronavirus, for example, the mankind respiratory disease and/or gastroenteritis and Hepatitis, gastroenteritis, encephalitis or respiratory disease in other animals.
Disease can be infective bronchitis (IB)；Pig epidemic diarrhea；Transmissible gastroenteritis；Mouse hepatitis virus； Pig blood cell agglutination encephalomyelitis；SARS (Severe Acute Respiratory Syndrome) (SARS)；Or blue comb.
Disease can be infective bronchitis.
For example, can by eye drops or intranasal administration by vaccine administration in the chicken of hatching.Although accurate, these methods It can be valuableness, such as large-scale broiler chicken group.Alternative solution includes spray inoculation or the application to drinking water, but uses this Class method is difficult to ensure uniform vaccine application.
Vaccine can be provided in the form for being suitable for its application, such as eye dropper for intraocularly using.
Vaccine can be applied by being inoculated in ovum, such as by injection embryonated egg.The advantage of ovum intradermal vaccine inoculation is it Early stage resistance to disease is provided.Different from spray inoculation and by drinking water application, it additionally aids the equal of each subject Even dosage application.
It can be by vaccine administration in any suitable compartment of egg, including allantoic fluid, yolk bag, amnion, gas room or embryo. It can be applied under shell (gas room) film and chorio-allantois.
Usually during the late stage of embryonic development by vaccine injection into embryonated egg, usually in the incubation period last four During/mono-, such as 3-4 days before hatching.In chicken, vaccine can between the 15-19 days of 21 day incubation period, such as It applies within 17th or 18 day.
The process can be used robot injection process automation, such as described in WO 2004/078203 those.
Vaccine can be applied together with other one or more vaccines, for example, being used for other diseases, such as newcastle disease virus (NDV) vaccine.The present invention also provides vaccine composition, it includes vaccine according to the present invention and it is one or more other Vaccine.The present invention also provides kits, and it includes vaccine according to the present invention and other one or more vaccines, for dividing It opens, serially or simultaneously apply.
Vaccine or vaccine composition of the invention can be used for treating people, animal or birds subject.For example, subject can To be chicken or mouse (such as laboratory mice, such as transgenic mice).
In general, doctor or animal doctor will determine the actual dose of most suitable individual subjects or subject group, and it will be with The age of particular subject, weight and reaction and change.
Optionally, composition may include pharmaceutical acceptable carrier, diluent, excipient or adjuvant.Pharmaceutical carrier, figuration Agent or the selection of diluent can be selected according to the administration method of intention and standard pharmaceutical practice.As carrier (or in addition to Outside carrier), pharmaceutical composition may include excipient or diluent, any suitable adhesive, lubricant, suspending agent, coating Agent, solubilizer and other potentially contribute to or increase the support agent of Viral delivery or immunogenicity.
The definition of term occurs throughout the specification.Before exemplary implementation scheme is more fully described, it should manage Solution, the present disclosure is not limited to described specific embodiments, therefore can of course change.It is also understood that art used herein Language is only used for the purpose of description specific embodiment, it is not intended to be restrictive, because the scope of the present disclosure is only by appended power The limitation of sharp claim.
Unless otherwise defined, otherwise all technical and scientific terms used herein have it is general with disclosure fields The logical identical meaning of the normally understood meaning of technical staff.Singleton,et al.,DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY,20ED.,John Wiley and Sons,New York(1994), And Hale&Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) general dictionary of many terms used in the disclosure is provided to technical staff.
The disclosure is not limited by illustrative methods disclosed herein and material, and with those of be described herein method and The similar or equivalent any method of material and material can be used for the embodiment practiced or test the disclosure.Digital scope includes The number of the range of definition.Unless otherwise stated, any nucleic acid sequence is write from left to right with 5 ' to 3 ' directions；Amino acid sequence Column are orientated with amino to carboxyl respectively from left to right to be write.
In the case where providing numberical range, it should be understood that unless the context clearly determines otherwise, otherwise also specific open Each median between the upper and lower bound of the range, until 1/10th of lower limit unit.It is any in the range Each smaller range between any other described or median in described value or median and the range is included in this In open.These small range of upper and lower bounds can be independently include within the scope of this or exclude in the range, and Being also covered by smaller range in present disclosure includes any, none or two limits each range, obeys the model Any limit clearly excluded in enclosing.In the case where the range includes one or two limitation, those limits for including are excluded The range of one or both of system is also included in the disclosure.
It must be noted that such as this paper and used in the attached claims, singular " one ", "/kind " " should/described " it include plural referents, unless the context is clearly stated.Thus, for example, refer to " enzyme " include it is a variety of this Class candidate agent and its equivalent well known by persons skilled in the art, etc..
As used herein, term "comprising" is synonymous with " comprising " or " containing ", and is inclusive or open, And it is not excluded for other, non-narration members, element or method and step.Term "comprising" also includes term " by ... group At ".
The present invention will be further described by embodiment now, these embodiments are intended for helping ordinary skill Personnel implement the present invention, and are not intended to limit the scope of the invention in any way.
Embodiment
Embodiment 1：The generation of IBV reverse genetics system based on M41-CK
By previously the vaccinia virus described in PCT/GB2010/001293 (being incorporated herein by reference) will reversely lose The Beaudette cDNA replacement passed in system is originated from the synthesis cDNA of M41 consensus sequence to generate M41-CK full-length cDNA.
By the way that 5 ' UTR-Nsp2-Nsp3 sequence of Beaudette is replaced with the corresponding sequence from IBV M41-CK into one Step modification is in Armesto, Cavanagh and Britton (2009) .PLoS ONE 4 (10):e7384.doi:10.1371/ IBV in vaccinia virus recombinant described in journal.pone.0007384 (rVV) rVV-BeauR-Rep-M41Struct CDNA, by the replicase from IBV Beaudette plants and structure and auxiliary gene and 3 ' UTR from IBV M41-CK Composition.Obtained IBV cDNA is by the 5 ' UTR-Nsp2-Nsp3 from M41, the Nsp4-Nsp16 from Beaudette and comes from The structure and auxiliary gene of M41 and 3 ' UTR composition.This is further modified by lacking Beaudette Nsp4-Nsp16 sequence cDNA.The cDNA for the shortage Nsp4-16 that modification obtains in four additional step, wherein sequential replace with of the Nsps of missing is come From the corresponding sequence of M41-CK, replaces cDNA and represent M41-CK Nsp4-8, Nsp9-12, Nsp12-14 and last Nsp15-16. Each replacement cDNA contains about 500 nucleotide for corresponding to 5 ' ends of 3 ' major part M41 sequences being previously inserted into and corresponds to About 500 nucleotide at 3 ' ends of M41S gene order.This allows to be inserted into M41cDNA sequence and sequential addition by homologous recombination Continuous M41 rdrp gene sequence.Using previously described instantaneous dominant selection (TDS) system of the present inventor (referring to PCT/ GB2010/001293), the synthesis cDNA containing Nsp sequence derived from M41 is added by homologous recombination.
Embodiment 2：Measurement recombinates the pathogenic of M41 virus
Three recombinants are generated using reverse genetics system described in embodiment 1.These recombinants are named as rIBV M41K-S-ADRP, rIBV M41K-A-ADRP and rIBV M41K-del3ab.
Show three kinds of recombinants grown in a manner of similar with wild type M41-CK/M41-K in Chicken kidney cell (Fig. 7 and 8)。
Nucleosides of the rIBV M41K-S-ADRP in adenosine diphosphate-region ribose -1 '-phosphatase (ADRP) of nsp-3 There is mutation at sour position G3685A, lead to the amino acid mutation from G to S.This mutation is introduced using plasmid shown in Fig. 1 In rVV containing M41-K.
RIBV M41K-A-ADRP has two at the nucleotide position A3664G and A3665C in the region ADRP of nsp-3 A mutation leads to the amino acid mutation from N to A.This is mutated using plasmid shown in Fig. 2 and introduces the rVV containing M41-K In.
RIBV M41K-del3ab has the missing from nucleotide position 23867 to 24214 in gene 3, causes to lack Auxilin 3a and 3b.The region is lacked from the rVV containing M41-K using plasmid shown in Fig. 3.
Three kinds of recombinant viruses are used to infect 8 age in days no-special pathogen (SPF) chickens by eye and rhinovaccination, to them Test it is pathogenic, as 3-7 days are observed by clinical sign daily after infecting, and test infect after the 4th day and the 6th day cilium live Property.The forfeiture of ciliary activity is the method for measuring the pathogenic perfect foundation of IBV.It was found that every kind in these recombinants is compared parent Viral M41-CK's is pathogenic low, as Figure 4-Figure 6.
Embodiment 3：Vaccine inoculation/Attack Research is carried out with M41-R
Under study for action test candidate vaccine virus, wherein fertilized eggs when (embryonation) occurs for embryo within 19 days Vaccine inoculation in ovum, and the wherein hatching rate of measurement inoculation egg.The clinical health of chicken is studied, and in 21 age in days with toxic The IB M41 challenge virus of power is with every dose 103.65EID50Attack chicken.
Clinical sign is studied after the attack protection by vaccine, and the progress ciliostasis test in 5 days after attack, to grind Study carefully the influence for the protection that challenge virus provides cilium movement and vaccine for ciliostasis (inhibiting ciliary movement).
Table 3
The result of hatching rate research
SPF broiler chicken egg is incubated for according to the conventional program that is incubated for.In incubation in 19 days, with every dose of IB vaccine strain IB M41-K-A- ADRP, IB M41-K-S-ADRP and IB M41-K-3ab are with 104EID50It is inoculated with 6 groups of 30 eggs.It is another with placebo, that is, PBS inoculation One group of 30 eggs.When hatching 21 days, egg incubation simultaneously obtains following result：
- PBS 87% (26/30) hatching, 1 chicken is dead after hatching, and 3 eggs holdings be not switched on and it is unmarked with It is injected for embryo.
- M41-K-A-ARDP 97% (29/30) hatching, the holding of 1 egg are not switched on and unmarked to inject for embryo.
- M41-K-S-ARDP 50% (15/30) hatching, 4 egg holdings are not switched on, wherein 1 label is used for embryo's note It penetrates.Other egg/animals fail to survive.
- M41-K-3ab 47% (18/30) hatching, 7 egg holdings are not switched on, wherein 3 labels are used for embryo's injection. Other egg/animals fail to survive.
IB M41-K-A-ADRP demonstrates the good hatching rate of inoculation egg, therefore is deemed appropriate to connect as ovum intradermal vaccine The vaccine of kind.It will be in attack test to this strain test efficacy, as detailed above.
All publications referred in description above are both incorporated herein by reference.Do not depart from the scope of the present invention and In the case where spirit, the various modifications and variations of the method for the invention and system be to those skilled in the art it is aobvious and It is clear to.Although having been combined specific preferred embodiment describes the present invention, it should be appreciated that, it is desirable that this hair of protection It is bright to be excessively limited to these specific embodiments.In fact, for the technology of molecular biology, virology or related fields The various modifications of the obvious mode for carrying out the present invention of personnel are intended to fall within the scope of the appended claims It is interior.
Sequence table
<110>Pierre Brett Institute
<120>Coronavirus
<130> P107816PCT
<150> GB 1601498.7
<151> 2016-01-27
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 27500
<212> DNA
<213>Avian infectious bronchitis virus
<400> 1
acttaagata gatattaata tatatctatc acactagcct tgcgctagat ttccaactta 60
acaaaacgga cttaaatacc tacagctggt cctcataggt gttccattgc agtgcacttt 120
agtgccctgg atggcacctg gccacctgtc aggtttttgt tattaaaatc ttattgttgc 180
tggtatcact gcttgttttg ccgtgtctca ctttatacat ccgttgcttg ggctacctag 240
tatccagcgt cctacgggcg ccgtggctgg ttcgagtgcg aagaacctct ggttcatcta 300
gcggtaggcg ggtgtgtgga agtagcactt cagacgtacc ggttctgttg tgtgaaatac 360
ggggtcacct ccccccacat acctctaagg gcttttgagc ctagcgttgg gctacgttct 420
cgcataaggt cggctatacg acgtttgtag ggggtagtgc caaacaaccc ctgaggtgac 480
aggttctggt ggtgtttagt gagcagacat acaatagaca gtgacaacat ggcttcaagc 540
ctaaaacagg gagtatctcc caaactaagg gatgtcattc ttgtatccaa agacattcct 600
gaacaacttt gtgacgcttt gtttttctat acgtcacaca accctaagga ttacgctgat 660
gcttttgcag ttaggcagaa gtttgatcgt aatctgcaga ctgggaaaca gttcaaattt 720
gaaactgtgt gtggtctctt cctcttgaag ggagttgaca aaataacacc tggcgtccca 780
gcaaaagtct taaaagccac ttctaagttg gcagatttag aagacatctt tggtgtctct 840
ccctttgcaa gaaaatatcg tgaacttttg aagacagcat gccagtggtc tcttactgta 900
gaaacactgg atgctcgtgc acaaactctt gatgaaattt ttgaccctac tgaaatactt 960
tggcttcagg tggcagcaaa aatccaagtt tcggctatgg cgatgcgcag gcttgttgga 1020
gaagtaactg caaaagtcat ggatgctttg ggctcaaata tgagtgctct tttccagatt 1080
tttaaacaac aaatagtcag aatttttcaa aaagcgctgg ctatttttga gaatgtgagt 1140
gaattaccac agcgtattgc agcacttaag atggcttttg ctaagtgtgc caagtccatt 1200
actgttgtgg ttatggagag gactctagtt gttagagagt tcgcaggaac ttgtcttgca 1260
agcattaatg gtgctgttgc aaaattcttt gaagaactcc caaatggttt catgggtgct 1320
aaaattttca ctacacttgc cttctttagg gaggctgcag tgaaaattgt ggataacata 1380
ccaaatgcac cgagaggcac taaagggttt gaagtcgttg gtaatgccaa aggtacacaa 1440
gttgttgtgc gtggcatgcg aaatgactta acactgcttg accaaaaagc tgaaattcct 1500
gtggagtcag aaggttggtc tgcaattttg ggtggacatc tttgctatgt ctttaagagt 1560
ggtgatcgct tttacgcggc acctctttca ggaaattttg cattgcatga tgtgcattgt 1620
tgtgagcgtg ttgtctgtct ttctgatggt gtaacaccgg agataaatga tggacttatt 1680
cttgcagcaa tctactcttc ttttagtgtc gcagaacttg tggcagccat taaaaggggt 1740
gaaccattta agtttctggg tcataaattt gtgtatgcaa aggatgcagc agtttctttt 1800
acattagcga aggctgctac tattgcagat gttttgaagc tgtttcaatc agcgcgtgtg 1860
aaagtagaag atgtttggtc ttcacttact gaaaagtctt ttgaattctg gaggcttgca 1920
tatggaaaag tgcgtaatct cgaagaattt gttaagactt gtttttgtaa ggctcaaatg 1980
gcgattgtga ttttagcgac agtgcttgga gagggcattt ggcatcttgt ttcgcaagtc 2040
atctataaag taggtggtct ttttactaaa gttgttgact tttgtgaaaa atattggaaa 2100
ggtttttgtg cacagttgaa aagagctaag ctcattgtca ctgaaaccct ctgtgttttg 2160
aaaggagttg cacagcattg ttttcaacta ttgctggatg caatacagtt tatgtataaa 2220
agttttaaga agtgtgcact tggtagaatc catggagact tgctcttctg gaaaggaggt 2280
gtgcacaaaa ttattcaaga gggcgatgaa atttggtttg acgccattga tagtattgat 2340
gttgaagatc tgggtgttgt tcaagaaaaa ttgattgatt ttgatgtttg tgataatgtg 2400
acacttccag agaaccaacc cggtcatatg gttcaaatcg aggatgacgg aaagaactac 2460
atgttcttcc gcttcaaaaa ggatgagaac atttattata caccaatgtc acagcttggt 2520
gctattaatg tggtttgcaa agcaggcggt aaaactgtca cctttggaga aactactgtg 2580
caagaaatac caccacctga tgttgtgttt attaaggtta gcattgagtg ttgtggtgaa 2640
ccatggaata caatcttcaa aaaggcttat aaggagccca ttgaagtaga gacagacctc 2700
acagttgaac aattgctctc tgtggtctat gagaaaatgt gtgatgatct caagctgttt 2760
ccggaggctc cagaaccacc accatttgag aatgtcacac ttgttgataa gaatggtaaa 2820
gatttggatt gcataaaatc atgccatctg atctatcgtg attatgagag cgatgatgac 2880
atcgaggaag aagatgcaga agaatgtgac acggattcag gtgatgctga ggagtgtgac 2940
actaattcag aatgtgaaga agaagatgag gatactaaag tgttggctct tatacaagac 3000
ccggcaagta acaaatatcc tctgcctctt gatgatgatt atagcgtcta caatggatgt 3060
attgttcata aggacgctct cgatgttgtg aatttaccat ctggtgaaga aacctttgtt 3120
gtcaataact gctttgaagg ggctgttaaa gctcttccgc agaaagttat tgatgttcta 3180
ggtgactggg gtgaggctgt tgatgcgcaa gaacaattgt gtcaacaaga atcaactcgg 3240
gtcatatctg agaaatcagt tgagggtttt actggtagtt gtgatgcaat ggctgaacaa 3300
gctattgttg aagagcagga aatagtacct gttgttgaac aaagtcagga tgtagttgtt 3360
tttacacctg cagacctaga agttgttaaa gaaacagcag aagaggttga tgagtttatt 3420
ctcatttctg ctgtccctaa agaagaagtt gtgtctcagg agaaagagga gccacaggtt 3480
gagcaagagc ctaccctagt tgttaaagca caacgtgaga agaaggctaa aaagttcaaa 3540
gttaaaccag ctacatgtga aaaacccaaa tttttggagt acaaaacatg tgtgggtgat 3600
ttggctgttg taattgccaa agcattggat gagtttaaag agttctgcat tgtaaacgct 3660
gcaaatgagc acatgtcgca tggtggtggc gttgcaaagg caattgcaga cttttgtgga 3720
ccggactttg ttgaatattg cgcggactat gttaagaaac atggtccaca gcaaaaactt 3780
gtcacacctt catttgttaa aggcattcaa tgtgtgaata atgttgtagg acctcgccat 3840
ggagacagca acttgcgtga gaagcttgtt gctgcttaca agagtgttct tgtaggtgga 3900
gtggttaact atgttgtgcc agttctctca tcagggattt ttggtgtaga ttttaaaata 3960
tcaatagatg ctatgcgcga agcttttaaa ggttgtgcca tacgcgttct tttattttct 4020
ctgagtcaag aacacatcga ttatttcgat gcaacttgta agcagaagac aatttatctt 4080
acggaggatg gtgttaaata ccgctctgtt gttttaaaac ctggtgattc tttgggtcaa 4140
tttggacagg tttttgcaag aaataaggta gtcttttcgg ctgatgatgt tgaggataaa 4200
gaaatcctct ttatacccac aactgacaag actattcttg aatattatgg tttagatgcg 4260
caaaagtatg taacatattt gcaaacgctt gcgcagaaat gggatgttca atatagagac 4320
aattttgtta tattagagtg gcgtgacgga aattgctgga ttagttcagc aatagttctc 4380
cttcaagctg ctaaaattag atttaaaggt tttcttgcag aagcatgggc taaactgttg 4440
ggtggagatc ctacagactt tgttgcctgg tgttatgcaa gttgcaatgc taaagtaggt 4500
gatttttcag atgctaattg gcttttggcc aatttagcag aacattttga cgcagattac 4560
acaaatgcac ttcttaagaa gtgtgtgtcg tgcaattgtg gtgttaagag ttatgaactt 4620
aggggtcttg aagcctgtat tcagccagtt cgagcaccta atcttctaca ttttaaaacg 4680
caatattcaa attgcccaac ctgtggtgca agtagtacgg atgaagtaat agaagcttca 4740
ttaccgtact tattgctttt tgctactgat ggtcctgcta cagttgattg tgatgaaaat 4800
gctgtaggga ctgttgtttt cattggctct actaatagtg gccattgtta tacacaagcc 4860
gatggtaagg cttttgacaa tcttgctaag gatagaaaat ttggaaggaa gtcgccttac 4920
attacagcaa tgtatacacg tttttctctt aggagtgaaa atcccctact tgttgttgaa 4980
catagtaagg gtaaagctaa agtagtaaaa gaagatgttt ctaaccttgc tactagttct 5040
aaagccagtt ttgacgatct tactgacttt gaacagtggt atgatagcaa catctatgag 5100
agtcttaaag tgcaggagac acctgataat cttgatgaat atgtgtcatt tacgacaaag 5160
gaagattcta agttgccact gacacttaaa gttagaggta tcaaatcagt tgttgacttt 5220
aggtctaagg atggttttac ttataagtta acacctgata ctgatgaaaa ttcaaaaaca 5280
ccagtctact acccagtctt ggattctatt agtcttaggg caatatgggt tgaaggcagt 5340
gctaattttg ttgttgggca tccaaattat tatagtaagt ctctccgaat tcccacgttt 5400
tgggaaaatg ccgagagctt tgttaaaatg ggttataaaa ttgatggtgt aactatgggc 5460
ctttggcgtg cagaacacct taataaacct aatttggaga gaatttttaa cattgctaag 5520
aaagctattg ttggatctag tgttgttact acgcagtgtg gtaaaatact agttaaagca 5580
gctacatacg ttgccgataa agtaggtgat ggtgtagttc gcaatattac agatagaatt 5640
aagggtcttt gtggattcac acgtggccat tttgaaaaga aaatgtccct acaatttcta 5700
aagacacttg tgttcttttt cttttatttc ttaaaggcta gtgctaagag tttagtttct 5760
agctataaga ttgtgttatg taaggtggtg tttgctacct tacttatagt gtggtttata 5820
tacacaagta atccagtagt gtttactgga atacgtgtgc tagacttcct atttgaaggt 5880
tctttatgtg gtccttataa tgactacggt aaagattctt ttgatgtgtt acgctattgt 5940
gcaggtgatt ttacttgtcg tgtgtgttta catgatagag attcacttca tctgtacaaa 6000
catgcttata gcgtagaaca aatttataag gatgcagctt ctggcattaa ctttaattgg 6060
aattggcttt atttggtctt tctaatatta tttgttaagc cagtggcagg ttttgttatt 6120
atttgttatt gtgttaagta tttggtattg agttcaactg tgttgcaaac tggtgtaggt 6180
tttctagatt ggtttgtaaa aacagttttt acccatttta attttatggg agcgggattt 6240
tatttctggc tcttttacaa gatatacgta caagtgcatc atatattgta ctgtaaggat 6300
gtaacatgtg aagtgtgcaa gagagttgca cgcagcaaca ggcaagaggt tagcgttgta 6360
gttggtggac gcaagcaaat agtgcatgtt tacactaatt ctggctataa cttttgtaag 6420
agacataatt ggtattgtag aaattgtgat gattatggtc accaaaatac atttatgtcc 6480
cctgaagttg ctggcgagct ttctgaaaag cttaagcgcc atgttaaacc tacagcatat 6540
gcttaccacg ttgtgtatga ggcatgcgtg gttgatgatt ttgttaattt aaaatataag 6600
gctgcaattc ctggtaagga taatgcatct tctgctgtta agtgtttcag tgttacagat 6660
tttttaaaga aagctgtttt tcttaaggag gcattgaaat gtgaacaaat atctaatgat 6720
ggttttatag tgtgtaatac acagagtgcg catgcactag aggaagcaaa gaatgcagcc 6780
gtctattatg cgcaatatct gtgtaagcca atacttatac ttgaccaggc actttatgag 6840
caattaatag tagagcctgt gtctaagagt gttatagata aagtgtgtag cattttgtct 6900
aatataatat ctgtagatac tgcagcttta aattataagg caggcacact tcgtgatgct 6960
ctgctttcta ttactaaaga cgaagaagcc gtagatatgg ctatcttctg ccacaatcat 7020
gaagtggaat acactggtga cggttttact aatgtgatac cgtcatatgg tatggacact 7080
gataagttga cacctcgtga tagagggttt ttgataaatg cagatgcttc tattgctaat 7140
ttaagagtca aaaatgctcc tccggtagta tggaagtttt ctgatcttat taaattgtct 7200
gacagttgcc ttaaatattt aatttcagct actgtcaagt caggaggtcg tttctttata 7260
acaaagtctg gtgctaaaca agttatttct tgtcataccc agaaactgtt ggtagagaaa 7320
aaggcaggtg gtgttattaa taacactttt aaatggttta tgagttgttt taaatggctt 7380
tttgtctttt atatactttt tacagcatgt tgtttgggtt actactatat ggagatgaat 7440
aaaagttttg ttcaccccat gtatgatgta aactccacac tgcatgttga agggttcaaa 7500
gttatagaca aaggtgttat tagagagatt gtgtcagaag ataattgttt ctctaataag 7560
tttgttaatt ttgacgcctt ttggggtaaa tcatatgaaa ataataaaaa ctgtccaatt 7620
gttacagttg ttatagatgg tgacgggaca gtagctgttg gtgttcctgg ttttgtatca 7680
tgggttatgg atggtgttat gtttgtgcat atgacacaga ctgatcgtag accttggtac 7740
attcctacct ggtttaatag agaaattgtt ggttacactc aggattcaat tatcactgag 7800
ggtagttttt atacatctat agcattattt tctgctagat gtttatattt aacagccagc 7860
aatacacctc aattgtattg ttttaatggc gacaatgatg cacctggagc cttaccattt 7920
ggtagtatta ttcctcatag agtatacttc caacctaatg gtgttaggct tatagttcca 7980
caacaaatac tgcatacacc ctacatagtg aagtttgttt cagacagcta ttgtagaggt 8040
agtgtatgtg agtatactaa accaggttac tgtgtgtcac tagactccca atgggttttg 8100
tttaatgatg aatacattag taaacctggc gttttctgtg gttctactgt tagagaactt 8160
atgtttaata tggttagtac attctttact ggtgtcaacc ctaatattta tattcagcta 8220
gcaactatgt ttttaatact agttgttatt gtgttaattt ttgcaatggt tataaagttt 8280
caaggtgttt ttaaagctta tgcgaccatt gtgtttacaa taatgttagt ttgggttatt 8340
aatgcatttg ttttgtgtgt acatagttat aatagtgttt tagctgttat attattagta 8400
ctctattgct atgcatcatt ggttacaagt cgcaatactg ctataataat gcattgttgg 8460
cttgttttta cctttggttt aatagtaccc acatggttgg cttgttgcta tctgggattt 8520
attctttata tgtacacacc gttggttttc tggtgttacg gtactactaa aaatactcgt 8580
aagttgtatg atggcaacga gtttgttggt aattatgacc ttgctgcgaa gagcactttt 8640
gttattcgtg gtactgaatt tgttaagctt acgaatgaga taggtgataa atttgaagcc 8700
tatctttctg cgtatgctag acttaaatac tattcaggca ctggtagtga gcaagattac 8760
ttgcaagctt gtcgtgcatg gttagcttat gctttggacc aatatagaaa tagtggtgtt 8820
gaggttgttt ataccccacc gcgttactct attggtgtta gtagactaca cgctggtttt 8880
aaaaaactag tttctcctag tagtgctgtt gagaagtgca ttgttagtgt ctcttataga 8940
ggcaataatc ttaatggact gtggctgggt gattctattt actgcccacg ccatgtgtta 9000
ggtaagttta gtggtgacca gtggggtgac gtactaaacc ttgctaataa tcatgagttt 9060
gaagttgtaa ctcaaaatgg tgttactttg aatgttgtca gcaggcggct taaaggagca 9120
gttttaattt tacaaactgc agttgccaat gctgaaactc ctaagtataa gtttgttaaa 9180
gctaattgtg gtgatagttt cactatagct tgttcttatg gtggtacagt tataggactt 9240
taccctgtca ctatgcgttc taatggtact attagagcat ctttcctagc aggagcctgt 9300
ggctcagttg gttttaatat agaaaagggt gtagttaatt tcttttatat gcaccatctt 9360
gagttaccta atgcattaca cactggaact gacctaatgg gtgagtttta tggtggttat 9420
gtagatgaag aggttgcgca aagagtgcca ccagataatc tagttactaa caatattgta 9480
gcatggctct atgcggcaat tattagtgtt aaagaaagta gtttttcaca acctaaatgg 9540
ttggagagta ctactgtttc tattgaagat tacaataggt gggctagtga taatggtttt 9600
actccatttt ccactagtac tgctattact aaattaagtg ctataactgg ggttgatgtt 9660
tgtaaactcc ttcgcactat tatggtaaaa agtgctcaat ggggtagtga tcccatttta 9720
ggacaatata attttgaaga cgaattgaca ccagaatctg tatttaatca agttggtggt 9780
gttaggttac agtcttcttt tgtaagaaaa gctacatctt ggttttggag tagatgtgta 9840
ttagcttgct tcttgtttgt gttgtgtgct attgtcttat ttacggcagt gccacttaag 9900
ttttatgtac atgcagctgt tattttgttg atggctgtgc tctttatttc ttttactgtt 9960
aaacatgtta tggcatacat ggacactttc ctattgccta cattgattac agttattatt 10020
ggagtttgtg ctgaagtccc tttcatatac aatactctaa ttagtcaagt tgttattttc 10080
ttaagccaat ggtatgatcc tgtagtcttt gatactatgg taccatggat gttattgcca 10140
ttagtgttgt acactgcttt taagtgtgta caaggctgct atatgaattc tttcaatact 10200
tctttgttaa tgctgtatca gtttatgaag ttaggttttg ttatttacac ctcttcaaac 10260
actcttactg catatacaga aggtaattgg gagttattct ttgagttggt tcacactatt 10320
gtgttggcta atgttagtag taattcctta attggtttaa ttgtttttaa gtgtgctaag 10380
tggattttat attattgcaa tgcaacatac tttaataatt atgtgttaat ggcagtcatg 10440
gttaatggca taggctggct ttgcacctgt tactttggat tgtattggtg ggttaataaa 10500
gtttttggtt taaccttagg taaatacaat tttaaagttt cagtagatca atataggtat 10560
atgtgtttgc ataaggtaaa tccacctaaa actgtgtggg aggtctttac tacaaatata 10620
cttatacaag gaattggagg cgatcgtgtg ttgcctatag ctacagtgca atctaaattg 10680
agtgatgtaa agtgtacaac tgttgtttta atgcagcttt tgactaagct taatgttgaa 10740
gcaaattcaa aaatgcatgc ttatcttgtt gagttacaca ataaaatcct cgcatctgat 10800
gatgttggag agtgcatgga taatttattg ggtatgctta taacactatt ttgtatagat 10860
tctactattg atttgggtga gtattgtgat gatatactta agaggtcaac tgtattacaa 10920
tcggttactc aagagttttc gcacataccc tcgtatgctg aatatgaaag agctaagagt 10980
atttatgaaa aggttttagc cgattctaaa aatggtggtg taacacagca agagcttgct 11040
gcatatcgta aagctgccaa tattgcaaag tcagtttttg atagagactt ggctgttcaa 11100
aagaagttag atagcatggc agaacgtgct atgacaacaa tgtataaaga ggcgcgtgta 11160
actgatagaa gagcaaaatt agtttcatca ttacatgcac tacttttttc aatgcttaag 11220
aaaatagatt ctgagaagct taatgtctta tttgaccagg cgaatagtgg tgttgtaccc 11280
ctagcaactg ttccaattgt ttgtagtaat aagcttaccc ttgttatacc agacccagag 11340
acgtgggtca agtgtgtgga gggtgtgcat gttacatatt caacagttgt ttggaatata 11400
gactgtgtta ctgatgccga tggcacagag ttacacccca cttctacagg tagtggattg 11460
acttactgta taagtggtga taatatagca tggcctttaa aggttaactt gactaggaat 11520
gggcataata aggttgatgt tgccttgcaa aataatgagc ttatgcctca cggtgtaaag 11580
acaaaggctt gcgtagcagg tgtagatcaa gcacattgta gcgttgagtc taaatgttat 11640
tatacaagta ttagtggcag ttcagttgta gctgctatta cctcttcaaa tcctaatctg 11700
aaagtagcct cttttttgaa tgaggcaggt aatcagattt atgtagactt agacccacca 11760
tgtaaatttg gtatgaaagt gggtgataag gttgaagttg tttacctgta ttttataaaa 11820
aatacgaggt ctattgtaag aggtatggta cttggtgcta tatctaatgt tgttgtgtta 11880
caatctaaag gtcatgagac agaggaagtg gatgctgtag gcattctctc actttgttct 11940
tttgcagtag atcctgcgga tacatattgt aaatatgtgg cagcaggtaa tcaaccttta 12000
ggtaactgtg ttaaaatgtt gacagtacat aatggtagtg gttttgcaat aacatcaaag 12060
ccaagtccaa ctccggatca ggattcttat ggaggagctt ctgtgtgtct ttattgtaga 12120
gcacatatag cacaccctgg cggagcagga aatttagatg gacgctgtca atttaaaggt 12180
tcttttgtgc aaatacctac tacggagaaa gatcctgttg gattctgtct acgtaacaag 12240
gtttgcactg tttgtcagtg ttggattggt tatggatgtc agtgtgattc acttagacaa 12300
cctaaacctt ctgttcagtc agttgctgtt gcatctggtt ttgataagaa ttatttaaac 12360
gggtacgggg tagcagtgag gctcggctga tacccctagc taatggatgt gaccccgatg 12420
ttgtaaagcg agcctttgat gtttgtaata aggaatcagc cggtatgttt caaaatttga 12480
agcgtaactg tgcacgattc caagaagtac gtgatactga agatggaaat cttgagtatt 12540
gtgattctta ttttgtggtt aaacaaacca ctcctagtaa ttatgaacat gagaaagctt 12600
gttatgaaga cttaaagtca gaagtaacag ctgatcatga tttctttgtg ttcaataaga 12660
acatttataa tattagtagg cagaggctta ctaagtatac tatgatggat ttttgctatg 12720
ctttgcggca ctttgaccca aaggattgcg aagttcttaa agaaatactt gtcacttatg 12780
gttgtataga agattatcac cctaagtggt ttgaagagaa taaggattgg tacgacccaa 12840
tagaaaaccc taaatattat gccatgttgg ctaaaatggg acctattgta cgacgtgctt 12900
tattgaatgc tattgagttc ggaaacctca tggttgaaaa aggttatgtt ggtgttatta 12960
cacttgataa ccaagatctt aatggcaaat tttatgattt tggtgatttt cagaagacag 13020
cgcctggtgc tggtgttcct gtttttgata cgtattattc ttacatgatg cccatcatag 13080
ccatgactga tgcgttggca cctgagaggt attttgaata tgatgtgcat aagggttata 13140
aatcttatga tctcctcaag tatgattata ctgaggagaa acaagatttg tttcagaagt 13200
actttaagta ttgggatcaa gagtatcacc ctaactgtcg cgactgtagt gatgacaggt 13260
gtttgataca ttgtgcaaac ttcaacatct tgttttctac acttgtaccg cagacttctt 13320
tcggtaattt gtgtagaaag gtttttgttg atggtgtacc atttatagct acttgtggct 13380
atcattctaa ggaacttggt gttattatga atcaagataa caccatgtca ttttcaaaaa 13440
tgggtttgag tcaactcatg cagtttgttg gagatcctgc cttgttagtg gggacatcca 13500
ataaattagt ggatcttaga acgtcttgtt ttagtgtttg tgctttagcg tctggtatta 13560
ctcatcaaac ggtaaaacca ggtcacttta acaaggattt ctacgatttt gcagagaagg 13620
ctggtatgtt taaggaaggt tcttctatac cacttaaaca tttcttctac ccacagactg 13680
gtaatgctgc tataaacgat tatgattatt atcgttataa caggcctacc atgtttgata 13740
tacgtcaact tttattttgt ttagaagtga cttctaaata ttttgaatgt tatgaaggcg 13800
gctgtatacc agcaagccaa gttgtagtta acaatttaga taagagtgca ggttatccgt 13860
tcaataagtt tggaaaggcc cgtctctatt atgaaatgag tctagaggag caggaccaac 13920
tctttgagag tacaaagaag aacgtcctgc ctactataac tcagatgaat ttaaaatatg 13980
ccatatccgc gaaaaataga gcgcgtacag tggcaggtgt gtctatcctt tctactatga 14040
ctaataggca gtttcatcag aagattctta agtctatagt caacactaga aacgctcctg 14100
tagttattgg aacaaccaag ttttatggcg gttgggataa catgttgaga aaccttattc 14160
agggtgttga agacccgatt cttatgggtt gggattatcc aaagtgtgat agagcaatgc 14220
ctaatttgtt gcgtatagca gcatctttag tactcgctcg taaacacact aattgttgta 14280
cttggtctga acgcgtttat aggttgtata atgaatgcgc tcaggtttta tctgaaactg 14340
tcttagctac aggtggtata tatgtgaaac ctggtggtac tagcagtgga gatgctacta 14400
ctgcttatgc aaacagtgtt ttcaacataa tacaagccac atctgctaat gttgcgcgtc 14460
ttttgagtgt tataacgcgt gatattgtat atgatgacat taagagcttg cagtatgaat 14520
tgtaccagca ggtttatagg cgagtcaatt ttgacccagc atttgttgaa aagttttatt 14580
cttatttgtg taagaatttc tcattgatga tcttgtctga cgacggtgtt gtttgttata 14640
acaacacatt agccaaacaa ggtcttgtag cagatatttc tggttttaga gaagttctct 14700
actatcagaa caatgttttt atggctgatt ctaaatgttg ggttgaacca gatttagaaa 14760
aaggcccaca tgaattttgt tcacagcaca caatgttagt ggaggttgat ggtgagccta 14820
gatacttgcc atatccagac ccatcacgta ttttgtgtgc atgtgttttt gtagatgatt 14880
tggataagac agaatctgtg gctgttatgg agcgttatat cgctcttgcc atagatgcgt 14940
acccactagt acatcatgaa aatgaggagt acaagaaggt attctttgtg cttctttcat 15000
acatcagaaa actctatcaa gagctttctc agaatatgct tatggactac tcttttgtaa 15060
tggatataga taagggtagt aaattttggg aacaggagtt ctatgaaaat atgtatagag 15120
cccctacaac attacagtct tgtggcgttt gtgtagtgtg taatagtcaa actatattgc 15180
gctgtggtaa ttgtattcgc aaaccatttt tgtgttgtaa gtgttgctat gaccatgtca 15240
tgcacacaga ccacaaaaat gttttgtcta taaatcctta catttgctca cagccaggtt 15300
gtggtgaagc agatgttact aaattgtacc tcggaggtat gtcatacttc tgcggtaatc 15360
ataaaccaaa gttatcaata ccgttagtat ctaatggtac agtgtttgga atttacaggg 15420
ctaattgtgc aggtagcgaa aatgttgatg attttaatca actagctact actaattggt 15480
ctactgtgga accttatatt ttggcaaatc gttgtgtaga ttcgttgaga cgctttgctg 15540
cagagacagt aaaagctaca gaagaattac ataagcaaca atttgctagt gcagaagtga 15600
gagaagtact ctcagatcgt gaattgattc tgtcttggga gccaggtaaa accaggcctc 15660
cattgaatag aaattatgtt ttcactggct ttcactttac tagaactagt aaagttcagc 15720
tcggtgattt tacatttgaa aaaggtgaag gtaaggacgt tgtctattat cgagcgacgt 15780
ctactgctaa attgtctgtt ggagacattt ttgttttaac ctcacacaat gttgtttctc 15840
ttatagcgcc aacgttgtgt cctcagcaaa ccttttctag gtttgtgaat ttaagaccta 15900
atgtgatggt acctgcgtgt tttgtaaata acattccatt gtaccattta gtaggcaagc 15960
agaagcgtac tacagtacaa ggccctcctg gcagtggtaa atcccatttt gctataggat 16020
tggcggctta ctttagtaac gcccgtgtcg tttttactgc atgctctcat gcagctgttg 16080
atgctttatg tgaaaaagct tttaagtttc ttaaagtaga tgattgcact cgtatagtac 16140
ctcaaaggac tactatcgat tgcttctcta agtttaaagc taatgacaca ggcaaaaagt 16200
acatttttag tactattaat gccttgccag aagttagttg tgacattctt ttggttgacg 16260
aggttagtat gttgaccaat tacgaattgt cttttattaa tggtaagata aactatcaat 16320
atgttgtgta tgtaggtgat cctgctcaat taccggcgcc tcgtacgttg cttaacggtt 16380
cactctctcc aaaggattat aatgttgtca caaaccttat ggtttgtgtt aaacctgaca 16440
ttttccttgc aaagtgttac cgttgtccta aagaaattgt agatactgtt tctactcttg 16500
tatatgatgg aaagtttatt gcaaataacc cggaatcacg tcagtgtttc aaggttatag 16560
ttaataatgg taattctgat gtaggacatg aaagtggctc agcctacaac ataactcaat 16620
tagaatttgt gaaagatttt gtctgtcgca ataaggaatg gcgggaagca acattcattt 16680
caccttataa tgctatgaac cagagagcct accgtatgct tggacttaat gttcagacag 16740
tagactcgtc tcaaggttcg gagtatgatt atgttatctt ttgtgttact gcagattcgc 16800
agcatgcact gaatattaac agattcaatg tagcgcttac aagagccaag cgtggtatac 16860
tagttgtcat gcgtcagcgt gatgaactat attcagctct taagtttata gagcttgata 16920
gtgtagcaag tctgcaaggt acaggcttgt ttaaaatttg caacaaagag tttagtggtg 16980
ttcacccagc ttatgcagtc acaactaagg ctcttgctgc aacttataaa gttaatgatg 17040
aacttgctgc acttgttaac gtggaagctg gttcagaaat aacatataaa catcttattt 17100
ctttgttagg gtttaagatg agtgttaatg ttgaaggctg ccacaacatg tttataacac 17160
gtgatgaggc tatccgcaac gtaagaggtt gggtaggttt tgatgtagaa gcaacacatg 17220
cttgcggtac taacattggt actaacctgc ctttccaagt aggtttctct actggtgcag 17280
actttgtagt tacgcctgag ggacttgtag atacttcaat aggcaataat tttgagcctg 17340
tgaattctaa agcacctcca ggtgaacaat ttaatcactt gagagcgtta ttcaaaagtg 17400
ctaaaccttg gcatgttgta aggccaagga ttgtgcaaat gttagcggat aacctgtgca 17460
acgtttcaga ttgtgtagtg tttgtcacgt ggtgtcatgg cctagaacta accactttgc 17520
gctattttgt taaaataggc aaggaccaag tttgttcttg cggttctaga gcaacaactt 17580
ttaattctca tactcaggct tatgcttgtt ggaagcattg cttgggtttt gattttgttt 17640
ataatccact cttagtggat attcaacagt ggggttattc tggtaaccta caatttaacc 17700
atgatttgca ttgtaatgtg catggacacg cacatgtagc ttctgcggat gctattatga 17760
cgcgttgtct tgcaattaat aatgcatttt gtcaagatgt caactgggat ttaacttacc 17820
ctcatatagc aaatgaggat gaagtcaatt ctagctgtag atatttacaa cgcatgtatc 17880
ttaatgcatg tgttgatgct cttaaagtta acgttgtcta tgatataggc aaccctaaag 17940
gtataaaatg tgttagacgt ggagacttaa attttagatt ctatgataag aatccaatag 18000
tacccaatgt caagcagttt gagtatgact ataatcagca caaagataag tttgctgatg 18060
gtctttgtat gttttggaat tgtaatgtgg attgttatcc cgacaattcc ttagtttgta 18120
ggtacgacac acgaaatttg agtgtgttta acctacctgg ttgtaatggt ggtagcttgt 18180
atgttaacaa gcatgcattc cacacaccta aatttgatcg cactagcttt cgtaatttga 18240
aagctatgcc attctttttc tatgactcat cgccttgcga gaccattcaa ttggatggag 18300
ttgcgcaaga ccttgtgtca ttagctacga aagattgtat cacaaaatgc aacataggcg 18360
gtgctgtttg taaaaagcac gcacaaatgt atgcagattt tgtgacttct tataatgcag 18420
ctgttactgc tggttttact ttttgggtta ctaataattt taacccatat aatttgtgga 18480
aaagtttttc agctctccag tctatcgaca atattgctta taatatgtat aagggtggtc 18540
attatgatgc tattgcagga gaaatgccca ctatcgtaac tggagataaa gtttttgtta 18600
tagatcaagg cgtagaaaaa gcagtttttt ttaatcaaac aattctgcct acatctgtag 18660
cgtttgagct gtatgcgaag agaaatattc gcacactgcc aaacaaccgt attttgaaag 18720
gtttgggtgt agatgtgact aatggatttg taatttggga ttacacgaac caaacaccac 18780
tataccgtaa tactgttaag gtatgtgcat atacagacat agaaccaaat ggcctaatag 18840
tgctgtatga tgatagatat ggtgattacc agtcttttct agctgctgat aatgctgttt 18900
tagtttctac acagtgttac aagcggtatt cgtatgtaga aataccgtca aacctgcttg 18960
ttcagaacgg tattccgtta aaagatggag cgaacctgta tgtttataag cgtgttaatg 19020
gtgcgtttgt tacgctacct aacacattaa acacacaggg tcgcagttat gaaacttttg 19080
aacctcgtag tgatgttgag cgtgattttc tcgacatgtc tgaggagagt tttgtagaaa 19140
agtatggtaa agaattaggt ctacagcaca tactgtatgg tgaagttgat aagccccaat 19200
taggtggttt acacactgtt ataggtatgt gcagactttt acgtgcgaat aagttgaacg 19260
caaagtctgt tactaattct gattctgatg tcatgcaaaa ttattttgta ttggcagaca 19320
atggttccta caagcaagtg tgtactgttg tggatttgct gcttgatgat ttcttagaac 19380
ttcttaggaa catactgaaa gagtatggta ctaataagtc taaagttgta acagtgtcaa 19440
ttgattacca tagcataaat tttatgactt ggtttgaaga tggcattatt aaaacatgtt 19500
atccacagct tcaatcagca tggacgtgtg gttataatat gcctgaactt tataaagttc 19560
agaattgtgt tatggaacct tgcaacattc ctaattatgg tgttggaata gcgttgccaa 19620
gtggtattat gatgaatgtg gcaaagtata cacaactctg tcaatacctt tcgaaaacaa 19680
caatgtgtgt accgcataat atgcgagtaa tgcattttgg agctggaagt gacaaaggag 19740
tggctccagg tagtactgtt cttaaacaat ggctcccaga agggacactc cttgtcgata 19800
atgatattgt agactatgtg tctgatgcac atgtttctgt gctttcagat tgcaataaat 19860
ataagacaga gcacaagttt gatcttgtga tatctgatat gtatacagac aatgattcaa 19920
aaagaaagca tgaaggcgtg atagccaata atggcaatga tgacgttttc atatatctct 19980
caagttttct tcgtaataat ttggctctag gtggtagttt tgctgtaaaa gtgacagaga 20040
caagttggca cgaagtttta tatgacattg cacaggattg tgcatggtgg acaatgtttt 20100
gtacagcagt gaatgcctct tcttcagaag cattcttggt tggtgttaat tatttgggtg 20160
caagtgaaaa ggttaaggtt agtggaaaaa cgctgcacgc aaattatata ttttggagga 20220
attgtaatta tttacaaacc tctgcttata gtatatttga cgttgctaag tttgatttga 20280
gattgaaagc aacaccagtt gttaatttga aaactgaaca aaagacagac ttagtcttta 20340
atttaattaa gtgtggtaag ttactggtaa gagatgttgg taacacctct tttactagtg 20400
actcttttgt gtgtactatg tagtgctgct ttgtatgaca gtagttctta cgtttactac 20460
taccaaagtg cctttagacc acctaatggt tggcatttac acgggggtgc ttatgcggta 20520
gttaatattt ctagcgaatc taataatgca ggctcttcac ctgggtgtat tgttggtact 20580
attcatggtg gtcgtgttgt taatgcttct tctatagcta tgacggcacc gtcatcaggt 20640
atggcttggt ctagcagtca gttttgtact gcacactgta acttttcaga tactacagtg 20700
tttgttacac attgttataa atatgatggg tgtcctataa ctggcatgct tcaaaagaat 20760
tttttacgtg tttctgctat gaaaaatggc cagcttttct ataatttaac agttagtgta 20820
gctaagtacc ctacttttaa atcatttcag tgtgttaata atttaacatc cgtatattta 20880
aatggtgatc ttgtttacac ctctaatgag accacagatg ttacatctgc aggtgtttat 20940
tttaaagctg gtggacctat aacttataaa gttatgagag aagttaaagc cctggcttat 21000
tttgttaatg gtactgcaca agatgttatt ttgtgtgatg gatcacctag aggcttgtta 21060
gcatgccagt ataatactgg caatttttca gatggctttt atccttttat taatagtagt 21120
ttagttaagc agaagtttat tgtctatcgt gaaaatagtg ttaatactac ttttacgtta 21180
cacaatttca cttttcataa tgagactggc gccaacccta atcctagtgg tgttcagaat 21240
attcaaactt accaaacaca aacagctcag agtggttatt ataattttaa tttttccttt 21300
ctgagtagtt ttgtttataa ggagtctaat tttatgtatg gatcttatca cccaagttgt 21360
aattttagac tagaaactat taataatggc ttgtggttta attcactttc agtttcaatt 21420
gcttacggtc ctcttcaagg tggttgcaag caatctgtct ttagtggtag agcaacttgt 21480
tgttatgctt attcatatgg aggtccttcg ctgtgtaaag gtgtttattc aggtgagtta 21540
gatcttaatt ttgaatgtgg actgttagtt tatgttacta agagcggtgg ctctcgtata 21600
caaacagcca ctgaaccgcc agttataact cgacacaatt ataataatat tactttaaat 21660
acttgtgttg attataatat atatggcaga actggccaag gttttattac taatgtaacc 21720
gactcagctg ttagttataa ttatctagca gacgcaggtt tggctatttt agatacatct 21780
ggttccatag acatctttgt tgtacaaggt gaatatggtc ttacttatta taaggttaac 21840
ccttgcgaag atgtcaacca gcagtttgta gtttctggtg gtaaattagt aggtattctt 21900
acttcacgta atgagactgg ttctcagctt cttgagaacc agttttacat taaaatcact 21960
aatggaacac gtcgttttag acgttctatt actgaaaatg ttgcaaattg cccttatgtt 22020
agttatggta agttttgtat aaaacctgat ggttcaattg ccacaatagt accaaaacaa 22080
ttggaacagt ttgtggcacc tttacttaat gttactgaaa atgtgctcat acctaacagt 22140
tttaatttaa ctgttacaga tgagtacata caaacgcgta tggataaggt ccaaattaat 22200
tgtctgcagt atgtttgtgg caattctctg gattgtagag atttgtttca acaatatggg 22260
cctgtttgtg acaacatatt gtctgtagta aatagtattg gtcaaaaaga agatatggaa 22320
cttttgaatt tctattcttc tactaaaccg gctggtttta atacaccatt tcttagtaat 22380
gttagcactg gtgagtttaa tatttctctt ctgttaacaa ctcctagtag tcctagaagg 22440
cgttctttta ttgaagacct tctatttaca agcgttgaat ctgttggatt accaacagat 22500
gacgcataca aaaattgcac tgcaggacct ttaggttttc ttaaggacct tgcgtgtgct 22560
cgtgaatata atggtttgct tgtgttgcct cccattataa cagcagaaat gcaaattttg 22620
tatactagtt ctctagtagc ttctatggct tttggtggta ttactgcagc tggtgctata 22680
ccttttgcca cacaactgca ggctagaatt aatcacttgg gtattaccca gtcacttttg 22740
ttgaagaatc aagaaaaaat tgctgcttcc tttaataagg ccattggtcg tatgcaggaa 22800
ggttttagaa gtacatctct agcattacaa caaattcaag atgttgttaa taagcagagt 22860
gctattctta ctgagactat ggcatcactt aataaaaatt ttggtgctat ttcttctatg 22920
attcaagaaa tctaccagca acttgacgcc atacaagcaa atgctcaagt ggatcgtctt 22980
ataactggta gattgtcatc actttctgtt ttagcatctg ctaagcaggc ggagcatatt 23040
agagtgtcac aacagcgtga gttagctact cagaaaatta atgagtgtgt taagtcacag 23100
tctattaggt actccttttg tggtaatgga cgacatgttc taaccatacc gcaaaatgca 23160
cctaatggta tagtgtttat acacttttct tatactccag atagttttgt taatgttact 23220
gcaatagtgg gtttttgtgt aaagccagct aatgctagtc agtatgcaat agtacccgct 23280
aatggtaggg gtatttttat acaagttaat ggtagttact acatcacagc acgagatatg 23340
tatatgccaa gagctattac tgcaggagat atagttacgc ttacttcttg tcaagcaaat 23400
tatgtaagtg taaataagac cgtcattact acattcgtag acaatgatga ttttgatttt 23460
aatgacgaat tgtcaaaatg gtggaatgac actaagcatg agctaccaga ctttgacaaa 23520
ttcaattaca cagtacctat acttgacatt gatagtgaaa ttgatcgtat tcaaggcgtt 23580
atacagggtc ttaatgactc tttaatagac cttgaaaaac tttcaatact caaaacttat 23640
attaagtggc cttggtatgt gtggttagcc atagcttttg ccactattat cttcatctta 23700
atactaggat gggttttctt catgactgga tgttgtggtt gttgttgtgg atgctttggc 23760
attatgcctc taatgagtaa gtgtggtaag aaatcttctt attacacgac ttttgataac 23820
gatgtggtaa cttaacaata cagacctaaa aagtctgttt aatgattcaa agtcccacgt 23880
ccttcctaat agtattaatt tttctttggt gtaaacttgt actaagttgt tttagagagt 23940
ttattatagc gctccaacaa ctaatacaag ttttactcca aattatcaat agtaacttac 24000
agcctagact gaccctttgt cacagtctag actaatgtta aacttagaag caattattga 24060
aactggtgag caagtgattc aaaaaatcag tttcaattta cagcatattt caagtgtatt 24120
aaacacagaa gtatttgacc cctttgacta ttgttattac agaggaggta atttttggga 24180
aatagagtca gctgaagatt gttcaggtga tgatgaattt attgaataag tcgctagagg 24240
aaaatggaag ttttctaaca gcgctttata tatttgtagg atttttagca ctttatcttc 24300
taggtagagc acttcaagca tttgtacagg ctgctgatgc ttgttgttta ttttggtata 24360
catgggtagt aattccagga gctaagggta cagcctttgt atataagtat acatatggta 24420
gaaaacttaa caatccggaa ttagaagcag ttattgtcaa cgagtttcct aagaacggtt 24480
ggaataataa aaatccagca aattttcaag atgtccaacg agacaaattg tactcttgac 24540
tttgaacagt cagttgagct ttttaaagag tataatttat ttataactgc attcttgttg 24600
ttcttaacca taatacttca gtatggctat gcaacaagaa gtaagtttat ttatatactg 24660
aaaatgatag tgttatggtg cttttggccc cttaacattg cagtaggtgt aatttcatgt 24720
atatacccac caaacacagg aggtcttgtc gcagcgataa tacttacagt gtttgcgtgt 24780
ctgtcttttg taggttattg gatccagagt attagactct ttaagcggtg taggtcatgg 24840
tggtcattta acccagaatc taatgccgta ggttcaatac tcctaactaa tggtcaacaa 24900
tgtaattttg ctatagagag tgtgccaatg gtgctttctc caattataaa gaatggtgtt 24960
ctttattgtg agggtcagtg gcttgctaag tgtgaaccag accacttgcc taaagatata 25020
tttgtttgta caccggatag acgtaatatc taccgtatgg tgcagaaata tactggtgac 25080
caaagcggaa ataagaaacg gtttgctacg tttgtctatg caaagcagtc agtagatact 25140
ggcgagctag aaagtgtagc aacaggaggg agtagtcttt acacctaaat gtgtgtgtgt 25200
agagagtatt taaaattatt ctttaatagt gcctctattt taagagcgca taatagtatt 25260
atttttgagg atattaatat aaatcctctc tgttttatac tctcttttca agagctatta 25320
tttaaaaaac agtttttcca ctcttttgtg ccaaaaacta ttgttgttaa tggtgtaacc 25380
tttcaagtag ataatggaaa agtctactac gaaggaaaac caatttttca gaaaggttgt 25440
tgtaggttgt ggttgagtta taaaaaagat taaactacct actacactta tttttataag 25500
aggcgtttta tcttacaagc gcttaataaa tacggacgat gaaatggctg actagttttg 25560
taagggcagt tatttcatgt tataaacccc tattattaac tcaattaaga gtattagata 25620
ggttaatctt agatcatgga ccaaaacaca tcttaacgtg tgttaggtgc gtgattttgt 25680
ttcaattaga tttagtttat aggttggcgt atacgcctac tcaatcgctg gtatgaataa 25740
tagtaaagat aatccttttt gcggagcaat agcaagaaaa gcgcgaattt atctgagaga 25800
aggattagat tgtgtttact ttcttaacaa agcaggacaa gcagagtctt gtcccgcgtg 25860
tacctctcta gtattccagg ggaaaacttg tgaggaacac aaatataata ataatctttt 25920
gtcatggcaa gcggtaaggc aactggaaag acagatgccc cagctccagt catcaaacta 25980
ggaggaccaa agccacctaa agttggttct tctggaaatg tatcttggtt tcaagcaata 26040
aaagccaaga agttaaattc acctccgcct aagtttgaag gtagcggtgt tcctgataat 26100
gaaaatctaa aaccaagtca gcagcatgga tattggagac gccaagctag gtttaagcca 26160
ggtaaaggtg gaagaaaacc agtcccagat gcttggtatt tttactatac tggaacagga 26220
ccagccgcta acctgaattg gggtgatagc caagatggta tagtgtgggt tgctggtaag 26280
ggtgctgata ctaaatttag atctaatcag ggtactcgtg actctgacaa gtttgaccaa 26340
tatccgctac ggttttcaga cggaggacct gatggtaatt tccgttggga tttcattcct 26400
ctgaatcgtg gcaggagtgg gagatcaaca gcagcttcat cagcagcatc tagtagagca 26460
ccatcacgtg aagtttcgcg tggtcgcagg agtggttctg aagatgatct tattgctcgt 26520
gcagcaagga taattcagga tcagcagaag aagggttctc gcattacaaa ggctaaggct 26580
gatgaaatgg ctcaccgccg gtattgcaag cgcactattc cacctaatta taaggttgat 26640
caagtgtttg gtccccgtac taaaggtaag gagggaaatt ttggtgatga caagatgaat 26700
gaggaaggta ttaaggatgg gcgcgttaca gcaatgctca acctagttcc tagcagccat 26760
gcttgtcttt tcggaagtag agtgacgccc agacttcaac cagatgggct gcacttgaaa 26820
tttgaattta ctactgtggt cccacgtgat gatccgcagt ttgataatta tgtaaaaatt 26880
tgtgatcagt gtgttgatgg tgtaggaaca cgtccaaaag atgatgaacc aagaccaaag 26940
tcacgctcaa gttcaagacc tgcaacaaga ggaaattctc cagcgccaag acagcagcgc 27000
cctaagaagg agaaaaagcc aaagaagcag gatgatgaag tggataaagc attgacctca 27060
gatgaggaga ggaacaatgc acagctggaa tttgatgatg aacccaaggt aattaactgg 27120
ggggattcag ccctaggaga gaatgaactt tgagtaaaat tcaatagtaa gagttaagga 27180
agataggcat gtagcttgat tacctacatg tctatcgcca gggaaatgtc taatttgtct 27240
acttagtagc ctggaaacga acggtagacc cttagatttt aatttagttt aatttttagt 27300
ttagtttaag ttagtttaga gtaggtataa agatgccagt gccggggcca cgcggagtac 27360
gaccgagggt acagcactag gacgcccatt aggggaagag ctaaatttta gtttaagtta 27420
agtttaattg gctatgtata gttaaaattt ataggctagt atagagttag agcaaaaaaa 27480
aaaaaaaaaa aaaaaaaaaa 27500
<210> 2
<211> 709
<212> DNA
<213>Avian infectious bronchitis virus
<400> 2
cttaacaata cagacctaaa aagtctgttt aatgattcaa agtcccacgt ccttcctaat 60
agtattaatt tttctttggt gtaaacttgt actaagttgt tttagagagt ttattatagc 120
gctccaacaa ctaatacaag ttttactcca aattatcaat agtaacttac agcctagact 180
gaccctttgt cacagtctag actaatgtta aacttagaag caattattga aactggtgag 240
caagtgattc aaaaaatcag tttcaattta cagcatattt caagtgtatt aaacacagaa 300
gtatttgacc cctttgacta ttgttattac agaggaggta atttttggga aatagagtca 360
gctgaagatt gttcaggtga tgatgaattt attgaataag tcgctagagg aaaatggaag 420
ttttctaaca gcgctttata tatttgtagg atttttagca ctttatcttc taggtagagc 480
acttcaagca tttgtacagg ctgctgatgc ttgttgttta ttttggtata catgggtagt 540
aattccagga gctaagggta cagcctttgt atataagtat acatatggta gaaaacttaa 600
caatccggaa ttagaagcag ttattgtcaa cgagtttcct aagaacggtt ggaataataa 660
aaatccagca aattttcaag atgtccaacg agacaaattg tactcttga 709
<210> 3
<211> 57
<212> PRT
<213>Avian infectious bronchitis virus
<400> 3
Met Ile Gln Ser Pro Thr Ser Phe Leu Ile Val Leu Ile Phe Leu Trp
1 5 10 15
Cys Lys Leu Val Leu Ser Cys Phe Arg Glu Phe Ile Ile Ala Leu Gln
20 25 30
Gln Leu Ile Gln Val Leu Leu Gln Ile Ile Asn Ser Asn Leu Gln Pro
35 40 45
Arg Leu Thr Leu Cys His Ser Leu Asp
50 55
<210> 4
<211> 64
<212> PRT
<213>Avian infectious bronchitis virus
<400> 4
Met Leu Asn Leu Glu Ala Ile Ile Glu Thr Gly Glu Gln Val Ile Gln
1 5 10 15
Lys Ile Ser Phe Asn Leu Gln His Ile Ser Ser Val Leu Asn Thr Glu
20 25 30
Val Phe Asp Pro Phe Asp Tyr Cys Tyr Tyr Arg Gly Gly Asn Phe Trp
35 40 45
Glu Ile Glu Ser Ala Glu Asp Cys Ser Gly Asp Asp Glu Phe Ile Glu
50 55 60
<210> 5
<211> 4782
<212> DNA
<213>Avian infectious bronchitis virus
<400> 5
ggtaaaactg tcacctttgg agaaactact gtgcaagaaa taccaccacc tgatgttgtg 60
tttattaagg ttagcattga gtgttgtggt gaaccatgga atacaatctt caaaaaggct 120
tataaggagc ccattgaagt agagacagac ctcacagttg aacaattgct ctctgtggtc 180
tatgagaaaa tgtgtgatga tctcaagctg tttccggagg ctccagaacc accaccattt 240
gagaatgtca cacttgttga taagaatggt aaagatttgg attgcataaa atcatgccat 300
ctgatctatc gtgattatga gagcgatgat gacatcgagg aagaagatgc agaagaatgt 360
gacacggatt caggtgatgc tgaggagtgt gacactaatt cagaatgtga agaagaagat 420
gaggatacta aagtgttggc tcttatacaa gacccggcaa gtaacaaata tcctctgcct 480
cttgatgatg attatagcgt ctacaatgga tgtattgttc ataaggacgc tctcgatgtt 540
gtgaatttac catctggtga agaaaccttt gttgtcaata actgctttga aggggctgtt 600
aaagctcttc cgcagaaagt tattgatgtt ctaggtgact ggggtgaggc tgttgatgcg 660
caagaacaat tgtgtcaaca agaatcaact cgggtcatat ctgagaaatc agttgagggt 720
tttactggta gttgtgatgc aatggctgaa caagctattg ttgaagagca ggaaatagta 780
cctgttgttg aacaaagtca ggatgtagtt gtttttacac ctgcagacct agaagttgtt 840
aaagaaacag cagaagaggt tgatgagttt attctcattt ctgctgtccc taaagaagaa 900
gttgtgtctc aggagaaaga ggagccacag gttgagcaag agcctaccct agttgttaaa 960
gcacaacgtg agaagaaggc taaaaagttc aaagttaaac cagctacatg tgaaaaaccc 1020
aaatttttgg agtacaaaac atgtgtgggt gatttggctg ttgtaattgc caaagcattg 1080
gatgagttta aagagttctg cattgtaaac gctgcaaatg agcacatgtc gcatggtggt 1140
ggcgttgcaa aggcaattgc agacttttgt ggaccggact ttgttgaata ttgcgcggac 1200
tatgttaaga aacatggtcc acagcaaaaa cttgtcacac cttcatttgt taaaggcatt 1260
caatgtgtga ataatgttgt aggacctcgc catggagaca gcaacttgcg tgagaagctt 1320
gttgctgctt acaagagtgt tcttgtaggt ggagtggtta actatgttgt gccagttctc 1380
tcatcaggga tttttggtgt agattttaaa atatcaatag atgctatgcg cgaagctttt 1440
aaaggttgtg ccatacgcgt tcttttattt tctctgagtc aagaacacat cgattatttc 1500
gatgcaactt gtaagcagaa gacaatttat cttacggagg atggtgttaa ataccgctct 1560
gttgttttaa aacctggtga ttctttgggt caatttggac aggtttttgc aagaaataag 1620
gtagtctttt cggctgatga tgttgaggat aaagaaatcc tctttatacc cacaactgac 1680
aagactattc ttgaatatta tggtttagat gcgcaaaagt atgtaacata tttgcaaacg 1740
cttgcgcaga aatgggatgt tcaatataga gacaattttg ttatattaga gtggcgtgac 1800
ggaaattgct ggattagttc agcaatagtt ctccttcaag ctgctaaaat tagatttaaa 1860
ggttttcttg cagaagcatg ggctaaactg ttgggtggag atcctacaga ctttgttgcc 1920
tggtgttatg caagttgcaa tgctaaagta ggtgattttt cagatgctaa ttggcttttg 1980
gccaatttag cagaacattt tgacgcagat tacacaaatg cacttcttaa gaagtgtgtg 2040
tcgtgcaatt gtggtgttaa gagttatgaa cttaggggtc ttgaagcctg tattcagcca 2100
gttcgagcac ctaatcttct acattttaaa acgcaatatt caaattgccc aacctgtggt 2160
gcaagtagta cggatgaagt aatagaagct tcattaccgt acttattgct ttttgctact 2220
gatggtcctg ctacagttga ttgtgatgaa aatgctgtag ggactgttgt tttcattggc 2280
tctactaata gtggccattg ttatacacaa gccgatggta aggcttttga caatcttgct 2340
aaggatagaa aatttggaag gaagtcgcct tacattacag caatgtatac acgtttttct 2400
cttaggagtg aaaatcccct acttgttgtt gaacatagta agggtaaagc taaagtagta 2460
aaagaagatg tttctaacct tgctactagt tctaaagcca gttttgacga tcttactgac 2520
tttgaacagt ggtatgatag caacatctat gagagtctta aagtgcagga gacacctgat 2580
aatcttgatg aatatgtgtc atttacgaca aaggaagatt ctaagttgcc actgacactt 2640
aaagttagag gtatcaaatc agttgttgac tttaggtcta aggatggttt tacttataag 2700
ttaacacctg atactgatga aaattcaaaa acaccagtct actacccagt cttggattct 2760
attagtctta gggcaatatg ggttgaaggc agtgctaatt ttgttgttgg gcatccaaat 2820
tattatagta agtctctccg aattcccacg ttttgggaaa atgccgagag ctttgttaaa 2880
atgggttata aaattgatgg tgtaactatg ggcctttggc gtgcagaaca ccttaataaa 2940
cctaatttgg agagaatttt taacattgct aagaaagcta ttgttggatc tagtgttgtt 3000
actacgcagt gtggtaaaat actagttaaa gcagctacat acgttgccga taaagtaggt 3060
gatggtgtag ttcgcaatat tacagataga attaagggtc tttgtggatt cacacgtggc 3120
cattttgaaa agaaaatgtc cctacaattt ctaaagacac ttgtgttctt tttcttttat 3180
ttcttaaagg ctagtgctaa gagtttagtt tctagctata agattgtgtt atgtaaggtg 3240
gtgtttgcta ccttacttat agtgtggttt atatacacaa gtaatccagt agtgtttact 3300
ggaatacgtg tgctagactt cctatttgaa ggttctttat gtggtcctta taatgactac 3360
ggtaaagatt cttttgatgt gttacgctat tgtgcaggtg attttacttg tcgtgtgtgt 3420
ttacatgata gagattcact tcatctgtac aaacatgctt atagcgtaga acaaatttat 3480
aaggatgcag cttctggcat taactttaat tggaattggc tttatttggt ctttctaata 3540
ttatttgtta agccagtggc aggttttgtt attatttgtt attgtgttaa gtatttggta 3600
ttgagttcaa ctgtgttgca aactggtgta ggttttctag attggtttgt aaaaacagtt 3660
tttacccatt ttaattttat gggagcggga ttttatttct ggctctttta caagatatac 3720
gtacaagtgc atcatatatt gtactgtaag gatgtaacat gtgaagtgtg caagagagtt 3780
gcacgcagca acaggcaaga ggttagcgtt gtagttggtg gacgcaagca aatagtgcat 3840
gtttacacta attctggcta taacttttgt aagagacata attggtattg tagaaattgt 3900
gatgattatg gtcaccaaaa tacatttatg tcccctgaag ttgctggcga gctttctgaa 3960
aagcttaagc gccatgttaa acctacagca tatgcttacc acgttgtgta tgaggcatgc 4020
gtggttgatg attttgttaa tttaaaatat aaggctgcaa ttcctggtaa ggataatgca 4080
tcttctgctg ttaagtgttt cagtgttaca gattttttaa agaaagctgt ttttcttaag 4140
gaggcattga aatgtgaaca aatatctaat gatggtttta tagtgtgtaa tacacagagt 4200
gcgcatgcac tagaggaagc aaagaatgca gccgtctatt atgcgcaata tctgtgtaag 4260
ccaatactta tacttgacca ggcactttat gagcaattaa tagtagagcc tgtgtctaag 4320
agtgttatag ataaagtgtg tagcattttg tctaatataa tatctgtaga tactgcagct 4380
ttaaattata aggcaggcac acttcgtgat gctctgcttt ctattactaa agacgaagaa 4440
gccgtagata tggctatctt ctgccacaat catgaagtgg aatacactgg tgacggtttt 4500
actaatgtga taccgtcata tggtatggac actgataagt tgacacctcg tgatagaggg 4560
tttttgataa atgcagatgc ttctattgct aatttaagag tcaaaaatgc tcctccggta 4620
gtatggaagt tttctgatct tattaaattg tctgacagtt gccttaaata tttaatttca 4680
gctactgtca agtcaggagg tcgtttcttt ataacaaagt ctggtgctaa acaagttatt 4740
tcttgtcata cccagaaact gttggtagag aaaaaggcag gt 4782
<210> 6
<211> 1594
<212> PRT
<213>Avian infectious bronchitis virus
<400> 6
Gly Lys Thr Val Thr Phe Gly Glu Thr Thr Val Gln Glu Ile Pro Pro
1 5 10 15
Pro Asp Val Val Phe Ile Lys Val Ser Ile Glu Cys Cys Gly Glu Pro
20 25 30
Trp Asn Thr Ile Phe Lys Lys Ala Tyr Lys Glu Pro Ile Glu Val Glu
35 40 45
Thr Asp Leu Thr Val Glu Gln Leu Leu Ser Val Val Tyr Glu Lys Met
50 55 60
Cys Asp Asp Leu Lys Leu Phe Pro Glu Ala Pro Glu Pro Pro Pro Phe
65 70 75 80
Glu Asn Val Thr Leu Val Asp Lys Asn Gly Lys Asp Leu Asp Cys Ile
85 90 95
Lys Ser Cys His Leu Ile Tyr Arg Asp Tyr Glu Ser Asp Asp Asp Ile
100 105 110
Glu Glu Glu Asp Ala Glu Glu Cys Asp Thr Asp Ser Gly Asp Ala Glu
115 120 125
Glu Cys Asp Thr Asn Ser Glu Cys Glu Glu Glu Asp Glu Asp Thr Lys
130 135 140
Val Leu Ala Leu Ile Gln Asp Pro Ala Ser Asn Lys Tyr Pro Leu Pro
145 150 155 160
Leu Asp Asp Asp Tyr Ser Val Tyr Asn Gly Cys Ile Val His Lys Asp
165 170 175
Ala Leu Asp Val Val Asn Leu Pro Ser Gly Glu Glu Thr Phe Val Val
180 185 190
Asn Asn Cys Phe Glu Gly Ala Val Lys Ala Leu Pro Gln Lys Val Ile
195 200 205
Asp Val Leu Gly Asp Trp Gly Glu Ala Val Asp Ala Gln Glu Gln Leu
210 215 220
Cys Gln Gln Glu Ser Thr Arg Val Ile Ser Glu Lys Ser Val Glu Gly
225 230 235 240
Phe Thr Gly Ser Cys Asp Ala Met Ala Glu Gln Ala Ile Val Glu Glu
245 250 255
Gln Glu Ile Val Pro Val Val Glu Gln Ser Gln Asp Val Val Val Phe
260 265 270
Thr Pro Ala Asp Leu Glu Val Val Lys Glu Thr Ala Glu Glu Val Asp
275 280 285
Glu Phe Ile Leu Ile Ser Ala Val Pro Lys Glu Glu Val Val Ser Gln
290 295 300
Glu Lys Glu Glu Pro Gln Val Glu Gln Glu Pro Thr Leu Val Val Lys
305 310 315 320
Ala Gln Arg Glu Lys Lys Ala Lys Lys Phe Lys Val Lys Pro Ala Thr
325 330 335
Cys Glu Lys Pro Lys Phe Leu Glu Tyr Lys Thr Cys Val Gly Asp Leu
340 345 350
Ala Val Val Ile Ala Lys Ala Leu Asp Glu Phe Lys Glu Phe Cys Ile
355 360 365
Val Asn Ala Ala Asn Glu His Met Ser His Gly Gly Gly Val Ala Lys
370 375 380
Ala Ile Ala Asp Phe Cys Gly Pro Asp Phe Val Glu Tyr Cys Ala Asp
385 390 395 400
Tyr Val Lys Lys His Gly Pro Gln Gln Lys Leu Val Thr Pro Ser Phe
405 410 415
Val Lys Gly Ile Gln Cys Val Asn Asn Val Val Gly Pro Arg His Gly
420 425 430
Asp Ser Asn Leu Arg Glu Lys Leu Val Ala Ala Tyr Lys Ser Val Leu
435 440 445
Val Gly Gly Val Val Asn Tyr Val Val Pro Val Leu Ser Ser Gly Ile
450 455 460
Phe Gly Val Asp Phe Lys Ile Ser Ile Asp Ala Met Arg Glu Ala Phe
465 470 475 480
Lys Gly Cys Ala Ile Arg Val Leu Leu Phe Ser Leu Ser Gln Glu His
485 490 495
Ile Asp Tyr Phe Asp Ala Thr Cys Lys Gln Lys Thr Ile Tyr Leu Thr
500 505 510
Glu Asp Gly Val Lys Tyr Arg Ser Val Val Leu Lys Pro Gly Asp Ser
515 520 525
Leu Gly Gln Phe Gly Gln Val Phe Ala Arg Asn Lys Val Val Phe Ser
530 535 540
Ala Asp Asp Val Glu Asp Lys Glu Ile Leu Phe Ile Pro Thr Thr Asp
545 550 555 560
Lys Thr Ile Leu Glu Tyr Tyr Gly Leu Asp Ala Gln Lys Tyr Val Thr
565 570 575
Tyr Leu Gln Thr Leu Ala Gln Lys Trp Asp Val Gln Tyr Arg Asp Asn
580 585 590
Phe Val Ile Leu Glu Trp Arg Asp Gly Asn Cys Trp Ile Ser Ser Ala
595 600 605
Ile Val Leu Leu Gln Ala Ala Lys Ile Arg Phe Lys Gly Phe Leu Ala
610 615 620
Glu Ala Trp Ala Lys Leu Leu Gly Gly Asp Pro Thr Asp Phe Val Ala
625 630 635 640
Trp Cys Tyr Ala Ser Cys Asn Ala Lys Val Gly Asp Phe Ser Asp Ala
645 650 655
Asn Trp Leu Leu Ala Asn Leu Ala Glu His Phe Asp Ala Asp Tyr Thr
660 665 670
Asn Ala Leu Leu Lys Lys Cys Val Ser Cys Asn Cys Gly Val Lys Ser
675 680 685
Tyr Glu Leu Arg Gly Leu Glu Ala Cys Ile Gln Pro Val Arg Ala Pro
690 695 700
Asn Leu Leu His Phe Lys Thr Gln Tyr Ser Asn Cys Pro Thr Cys Gly
705 710 715 720
Ala Ser Ser Thr Asp Glu Val Ile Glu Ala Ser Leu Pro Tyr Leu Leu
725 730 735
Leu Phe Ala Thr Asp Gly Pro Ala Thr Val Asp Cys Asp Glu Asn Ala
740 745 750
Val Gly Thr Val Val Phe Ile Gly Ser Thr Asn Ser Gly His Cys Tyr
755 760 765
Thr Gln Ala Asp Gly Lys Ala Phe Asp Asn Leu Ala Lys Asp Arg Lys
770 775 780
Phe Gly Arg Lys Ser Pro Tyr Ile Thr Ala Met Tyr Thr Arg Phe Ser
785 790 795 800
Leu Arg Ser Glu Asn Pro Leu Leu Val Val Glu His Ser Lys Gly Lys
805 810 815
Ala Lys Val Val Lys Glu Asp Val Ser Asn Leu Ala Thr Ser Ser Lys
820 825 830
Ala Ser Phe Asp Asp Leu Thr Asp Phe Glu Gln Trp Tyr Asp Ser Asn
835 840 845
Ile Tyr Glu Ser Leu Lys Val Gln Glu Thr Pro Asp Asn Leu Asp Glu
850 855 860
Tyr Val Ser Phe Thr Thr Lys Glu Asp Ser Lys Leu Pro Leu Thr Leu
865 870 875 880
Lys Val Arg Gly Ile Lys Ser Val Val Asp Phe Arg Ser Lys Asp Gly
885 890 895
Phe Thr Tyr Lys Leu Thr Pro Asp Thr Asp Glu Asn Ser Lys Thr Pro
900 905 910
Val Tyr Tyr Pro Val Leu Asp Ser Ile Ser Leu Arg Ala Ile Trp Val
915 920 925
Glu Gly Ser Ala Asn Phe Val Val Gly His Pro Asn Tyr Tyr Ser Lys
930 935 940
Ser Leu Arg Ile Pro Thr Phe Trp Glu Asn Ala Glu Ser Phe Val Lys
945 950 955 960
Met Gly Tyr Lys Ile Asp Gly Val Thr Met Gly Leu Trp Arg Ala Glu
965 970 975
His Leu Asn Lys Pro Asn Leu Glu Arg Ile Phe Asn Ile Ala Lys Lys
980 985 990
Ala Ile Val Gly Ser Ser Val Val Thr Thr Gln Cys Gly Lys Ile Leu
995 1000 1005
Val Lys Ala Ala Thr Tyr Val Ala Asp Lys Val Gly Asp Gly Val
1010 1015 1020
Val Arg Asn Ile Thr Asp Arg Ile Lys Gly Leu Cys Gly Phe Thr
1025 1030 1035
Arg Gly His Phe Glu Lys Lys Met Ser Leu Gln Phe Leu Lys Thr
1040 1045 1050
Leu Val Phe Phe Phe Phe Tyr Phe Leu Lys Ala Ser Ala Lys Ser
1055 1060 1065
Leu Val Ser Ser Tyr Lys Ile Val Leu Cys Lys Val Val Phe Ala
1070 1075 1080
Thr Leu Leu Ile Val Trp Phe Ile Tyr Thr Ser Asn Pro Val Val
1085 1090 1095
Phe Thr Gly Ile Arg Val Leu Asp Phe Leu Phe Glu Gly Ser Leu
1100 1105 1110
Cys Gly Pro Tyr Asn Asp Tyr Gly Lys Asp Ser Phe Asp Val Leu
1115 1120 1125
Arg Tyr Cys Ala Gly Asp Phe Thr Cys Arg Val Cys Leu His Asp
1130 1135 1140
Arg Asp Ser Leu His Leu Tyr Lys His Ala Tyr Ser Val Glu Gln
1145 1150 1155
Ile Tyr Lys Asp Ala Ala Ser Gly Ile Asn Phe Asn Trp Asn Trp
1160 1165 1170
Leu Tyr Leu Val Phe Leu Ile Leu Phe Val Lys Pro Val Ala Gly
1175 1180 1185
Phe Val Ile Ile Cys Tyr Cys Val Lys Tyr Leu Val Leu Ser Ser
1190 1195 1200
Thr Val Leu Gln Thr Gly Val Gly Phe Leu Asp Trp Phe Val Lys
1205 1210 1215
Thr Val Phe Thr His Phe Asn Phe Met Gly Ala Gly Phe Tyr Phe
1220 1225 1230
Trp Leu Phe Tyr Lys Ile Tyr Val Gln Val His His Ile Leu Tyr
1235 1240 1245
Cys Lys Asp Val Thr Cys Glu Val Cys Lys Arg Val Ala Arg Ser
1250 1255 1260
Asn Arg Gln Glu Val Ser Val Val Val Gly Gly Arg Lys Gln Ile
1265 1270 1275
Val His Val Tyr Thr Asn Ser Gly Tyr Asn Phe Cys Lys Arg His
1280 1285 1290
Asn Trp Tyr Cys Arg Asn Cys Asp Asp Tyr Gly His Gln Asn Thr
1295 1300 1305
Phe Met Ser Pro Glu Val Ala Gly Glu Leu Ser Glu Lys Leu Lys
1310 1315 1320
Arg His Val Lys Pro Thr Ala Tyr Ala Tyr His Val Val Tyr Glu
1325 1330 1335
Ala Cys Val Val Asp Asp Phe Val Asn Leu Lys Tyr Lys Ala Ala
1340 1345 1350
Ile Pro Gly Lys Asp Asn Ala Ser Ser Ala Val Lys Cys Phe Ser
1355 1360 1365
Val Thr Asp Phe Leu Lys Lys Ala Val Phe Leu Lys Glu Ala Leu
1370 1375 1380
Lys Cys Glu Gln Ile Ser Asn Asp Gly Phe Ile Val Cys Asn Thr
1385 1390 1395
Gln Ser Ala His Ala Leu Glu Glu Ala Lys Asn Ala Ala Val Tyr
1400 1405 1410
Tyr Ala Gln Tyr Leu Cys Lys Pro Ile Leu Ile Leu Asp Gln Ala
1415 1420 1425
Leu Tyr Glu Gln Leu Ile Val Glu Pro Val Ser Lys Ser Val Ile
1430 1435 1440
Asp Lys Val Cys Ser Ile Leu Ser Asn Ile Ile Ser Val Asp Thr
1445 1450 1455
Ala Ala Leu Asn Tyr Lys Ala Gly Thr Leu Arg Asp Ala Leu Leu
1460 1465 1470
Ser Ile Thr Lys Asp Glu Glu Ala Val Asp Met Ala Ile Phe Cys
1475 1480 1485
His Asn His Glu Val Glu Tyr Thr Gly Asp Gly Phe Thr Asn Val
1490 1495 1500
Ile Pro Ser Tyr Gly Met Asp Thr Asp Lys Leu Thr Pro Arg Asp
1505 1510 1515
Arg Gly Phe Leu Ile Asn Ala Asp Ala Ser Ile Ala Asn Leu Arg
1520 1525 1530
Val Lys Asn Ala Pro Pro Val Val Trp Lys Phe Ser Asp Leu Ile
1535 1540 1545
Lys Leu Ser Asp Ser Cys Leu Lys Tyr Leu Ile Ser Ala Thr Val
1550 1555 1560
Lys Ser Gly Gly Arg Phe Phe Ile Thr Lys Ser Gly Ala Lys Gln
1565 1570 1575
Val Ile Ser Cys His Thr Gln Lys Leu Leu Val Glu Lys Lys Ala
1580 1585 1590
Gly

Claims (27)

1. the coronavirus of attenuation living, it includes in nonstructural proteins nsp-3 mutation and/or auxiliary protein 3a and/ Or the missing of 3b.
2. coronavirus according to claim 1, wherein the mutation in the nsp-3 is located at adenosine diphosphate-ribose -1 '-phosphorus Sour enzyme (ADRP) region.
3. coronavirus according to claim 1 or 2, wherein nsp-3 gene coding includes one selected from the group below Or the protein of multiple amino acid mutations：
a)SEQ ID NO:373rd Asn (N) to Ala (A) in 6；With
b)SEQ ID NO:379th Gly (G) to Ser (S) in 6.
4. coronavirus according to any one of the preceding claims, wherein nsp-3 gene coding includes SEQ ID NO:The 373rd amino acids are mutated the protein of Asn (N) to Ala (A) in 6.
5. coronavirus as claimed in one of claims 1-3, wherein nsp-3 gene coding includes SEQ ID NO:6 In the 379th amino acids mutation Gly (G) to Ser (S) protein.
6. coronavirus according to any one of the preceding claims, wherein the nsp-3 gene includes selected from the group below One or more nucleotide replace：
A) with SEQ ID NO:Sequence shown in 5 is compared to the A at the A to G and nucleotide position 1117 at nucleotide position 1116 To C；With
B) with SEQ ID NO:Sequence shown in 5 is compared to the G to A at nucleotide position 1138.
7. coronavirus according to claim 1, it includes SEQ ID NO:The nucleotide position 37 of sequence shown in 2 to 384 missing.
8. coronavirus according to any one of the preceding claims is infectious bronchitis virus (IBV).
9. coronavirus according to any one of the preceding claims is IBV M41.
10. coronavirus according to claim 9, it includes S protein, wherein the S protein at least partially from removing IBV serotype except M41.
11. coronavirus according to claim 10, wherein S1 subunit is from the IBV serotype in addition to M41.
12. coronavirus according to any one of the preceding claims, having compared with wild type coronavirus reduces It is pathogenic so that when the virus is applied to embryonated egg (embryonated egg), can replicate and not to embryo It is pathogenic.
13. the modified nsp-3 gene as defined in any one of claims 1 to 6.
14. by the protein of modified nsp-3 gene coding according to claim 13.
15. plasmid, it includes modified nsp-3 gene according to claim 13 or with auxilin 3a and/or 3b The modified gene 3 of missing.
16. the method for being used to prepare coronavirus according to any one of claim 1 to 12 comprising following steps：
(i) plasmid transfection according to claim 15 is entered in host cell；
(ii) host cell described in recombinant virus infection, the recombinant virus include with nsp-3 gene and/or gene 3 The genome of coronavirus strain；
(iii) allow to occur between the corresponding sequence in gene order and the recombinant virus genomes in the plasmid homologous Recombination is to generate modified gene；And
(iv) selection includes the recombinant virus of the modified gene.
17. according to the method for claim 16, wherein the recombinant virus is vaccinia virus.
18. method according to claim 16 or 17, further comprising the steps of：
(v) recombination coronavirus of the recycling comprising the modified gene from the DNA of the recombinant virus from step (iv).
19. the cell of coronavirus according to any one of claims 1 to 12 can be generated.
20. vaccine, it includes coronavirus according to any one of claims 1 to 12 and pharmaceutical acceptable carriers.
21. the method for treating and/or preventing the disease in subject, the method includes applying right to the subject It is required that the step of 20 vaccine.
22. vaccine according to claim 20 is used to treat and/or prevent the disease in subject.
23. coronavirus according to any one of claims 1 to 12 is in preparation for treating and/or preventing the disease in subject Purposes in the vaccine of disease.
24. according to method, vaccine or the purposes of claim 21,22 or 23, wherein the disease is infective bronchitis (IB)。
25. according to the method for claim 21, wherein the method for administration is selected from the group：Eye drops application, intranasal administration, It is injected in injection and ovum after drinking water application, hatching.
26. according to the method for claim 24, wherein the vaccine inoculation is the inoculation of ovum intradermal vaccine.
27. the method for being used to prepare vaccine according to claim 20 comprising with according to any one of claims 1 to 12 The step of cell of coronavirus infection claim 19.
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